Embryonic Stem Cell-Like Cells

ABSTRACT

Disclosed is a method for preparing an embryonic stem cell (ESC)-like cell, which includes the steps of: (a) obtaining a first cell population from a mammalian tissue or body fluid, wherein the first cell population comprises adult stem cells; (b) obtaining a second somatic cell population from a mammalian tissue, wherein the mammalian tissue is different from the mammalian tissue in step (a) and the second cell population is different from the first cell population; (c) coculturing the first cell population and the second cell population in a medium for a period of time sufficient to form a colony from either the first cell population or the second cell population; and (d) subculturing a cell from the colony in a medium for a period time sufficient to prepare the ESC-like cell.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for preparing an embryonicstem cell-like cell having pluripotency, and a mammalian tissue-derivedembryonic stem cell-like cell.

2. Description of the Related Art

Although patient-specific somatic cell nuclear transfer (SCNT) is anabsolute method for developing an immune reaction-free cell therapy,several methods have been suggested for replacing SCNT to avoid humancloning¹⁻¹⁰. However, each alternative suggested has various limitationsfor developing as the patient-specific therapy. Oocyte parthenogenesisis one possible alternative. Embryonic stem (ES) cells have been derivedfrom the parthenogenesis of ovulated oocytes in primates (Vrana, K. E.et al. Nonhuman primate parthenogenetic stem cells. Proc. Natl. Acad.Sci. USA 100, 11911-11916 (2003)) and recently in human (Brevini et al.,ESHRE annual meeting in 2006). We have established autologous ES cellsvia the parthenogenesis of immature oocytes collected from preantralfollicles (unpublished data, PCT/KR2006/001891). However, these methodsdo not eliminate completely the need for human cloning.

Throughout this application, various publications and patents arereferenced and citations are provided in parentheses. The disclosures ofthese publications and patents in their entities are hereby incorporatedby references into this application in order to more fully describe thisinvention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

Under such circumstances, the present inventors have made intensiveresearches to meet long-felt need in the art, and as a result, developeda novel method for successfully preparing an embryonic stem cell(ESC)-like cell having pluripotency without embryos and gametes.

Accordingly, it is an object of this invention to provide a method forpreparing an embryonic stem cell (ESC)-like cell.

It is another object of this invention to provide a mammalian tissue orbody fluid-derived embryonic stem cell (ESC)-like cell.

It is still another object of this invention to provide a culture mediumfor dedifferentiating a mammalian cell having no pluripotency into anembryonic stem cell (ESC)-like cell having pluripotency.

It is further object of this invention to provide a culture medium forproducing an embryonic stem cell (ESC)-like cell from a mammalian tissuecell.

Other objects and advantages of the present invention will becomeapparent from the detailed description to follow and together with theappended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. In situ hybridization of ovarian tissue for the detection ofOct-4 and Nanog mRNA expression, and double immunostaining ofOct-4-positive ovarian tissue-dissociated cells for the Oct-4, Vasa,AMH, and CD44 markers of stem cells, germ cells, follicular cells, andmesenchymal stem cells, respectively. (a) In situ hybridization ofovaries retrieved from an 8-week-old, adult mouse (B6CBAF1;C57BL/6×CBA/Ca) with Oct-4- and Nanog-specific mRNA probes. Oct-4 andNanog mRNA expression is evident in the numerous ovarian medullae nearthe blood vessel (OM). The theca cell region of different stage ovarianfollicles (OF) also show the mRNA expression. (CL) corpus luteum. Scalebar=100 μm. (b) The ovarian tissue cells immediately after dissociationwere double-immunostained. The Oct-4-positive cells are concomitantlyimmunostained with anti-Nanog, anti-Vasa or anti-CD44 antibodies, whilethey are not positive for AMH staining (confocal microscope image).Phase-contrast image of Oct-4-positive plus Nanog-, Vasa- orCD44-positive cells and of AMH-positive, but Oct-4 negative cell(arrows). Scale bar=10 μm.

FIG. 2. Morphology of embryonic stem cell (ESC)-like cells derived bythe coculturing of adult ovarian cells and embryonic fibroblasts. (a)Colony-forming ESCs of the E14 cell line. (b) Colony-forming ESC-likecells on day 7 of primary culture. (c) after ten subpassages (day 37 ofculture) and (d) after 50 subpassages (day 157 of culture). Scale bar=50μm.

FIG. 3. Immunostaining with anti-Oct-4 (embryonic stem cell-specific),anti-Nanog (embryonic stem cell-specific), anti-Vasa (germcell-specific), anti-Fragilis (germ cell-specific), anti-CD44(mesenchymal stem cell-specific) or anti-AMH (follicular cell-specific)of B6D2F1 (C57BL/6×DBA2; a, c) and ICR (b, d) mouse embryonicfibroblasts (MEFs) with (c, d) or without (a, b) mitomycin C treatment.MEFs of each strain were collected from 13.5-day-old fetuses afterremoval of the internal organs, head, and extremities. Immunostaining ofMEFs formed confluent monolayer was conducted. Confluent monolayerformation is detected, but none of the cells are positive for any of themarkers tested. Scale bar=100 μm.

FIG. 4. Karyotyping of dissociated ovarian cells, established embryonicstem cell (ESC)-like cells and mouse embryonic fibroblast (MEF).Air-dried chromosome preparations were conducted using Cytovision andflow cytometry was used for effective karyotyping large population ofcells. PCR analysis was further conducted using the Xchromosome-specific Xist and Y chromosome-specific Zfy primers. Thecells were immediately dissociated from the ovarian tissue (a, b) andMEF (g), and colony-forming cells (tScB6CD-SNU-1) were collected fromearly (6-9 passages; c, d) and late (30 passages; e, f) passages. Thedissociated cells have diploid chromosomes, while the ESC-like cellscollected from the early and late passages have tetraploid chromosomes.(h) PCR analysis of late-passage colony-forming cells. Lane 1, E14 cellswith n lane 2, established ScB6CD-SNU-1; lane 3, ScB6CD-SNU-2 line; lane4, established ScBCD-SNU-3 line; lane 5, established ScB6CD-SNU-4 line;lane 6, established ScB6CD-SNU-5 line; lane 7, established ScB6CD-SNU-6line. The established ESC-like cells of all six lines express the Xchromosome-specific Xist gene but not the Zfy gene.

FIG. 5 a-5 b. Characterization of embryonic stem cell (ESC)-like cellsderived from the coculturing of F1 (B6D2F1; C57BL/6×DBA2) embryonicfibroblasts and adult F1 (B6CBAF1; C57BL/6×CBA/Ca) ovarian cells: FIG. 5a. Similar to the E14 ES cell line, the ESC-like cells are positive forSSEA-1, Oct-4, integrin α6, integrin and AP. However, both the E14 EScells and the established cells are negative for SSEA-3 and the SSEA-4.Scale bar=50 μm. FIG. 5 b. Both cell types express the pluripotentcell-specific Oct-4, Nanog, Rex-1, Cripto, Dnmt3b, Tert, Lif Rc, Stat3,Bmp4, Foxd3, Sox2, CD9, and Gdf3 genes. +, Reverse transcription usingisolated total RNA, −, Non-reverse transcription using isolated totalRNA.

FIG. 6. Telomerase activities of the established embryonic stem cell(ESC)-like cells detected by the telomeric repeat amplication protocolassay. Six cell lines were evaluated. The ladder of telomerase productsamplified by PCR was shown with six-base increments starting at 50nucleotides at the portion indicated by the asterisk. All of the celllines express high levels of telomerase activity. Lane 1, positivecontrol (E14 embryonic stem cells); lane 2, MEFs; lane 3, PCR controlwithout the addition of template; lanes 4-9, colony-forming cells oftScB6CD-SNU-1, tScB6CD-SNU-2, tScB6CD-SNU-3, tScB6CD-SNU-4,tScB6CD-SNU-5, and tScB6CD-SNU-6, respectively.

FIG. 7. Characterization of embryonic stem cell (ESC)-like cells byfluorescence activated cell sorting (FACS) analysis using mesenchymalstem cell (MSC)-specific marker CD44 (a) and Sca-1 (b), epithelial stemcell-specific marker CD34 (c) and hematopoietic stem cell-specificmarker CD45 (d). The red color represents peaks of MSCs used as thecontrol. The E14 ES and established ESC-like cells were all negative foranti-CD44, anti-Sca-1, anti-CD34 and anti-CD45 antibodies.

FIG. 8. Immunostaining of embryonic stem cell (ESC)-like cells with theVasa and Fragilis germ cell-specific markers and with the follicularcell-specific marker AMH. All six lines of established ESC-like cellswere analyzed. The colony-forming cells retrieved at the eighthsubpassage are not positive for Vasa (a), Fragilis (b), and AMH (c).Scale bar=50 μm (a, b) and 100 μm (c).

FIG. 9 a-9 b. In vitro differentiation of embryonic stem cell (ESC)-likecells derived from the coculturing of F1 (B6D2F1; C57BL/6×DBA2)embryonic fibroblasts and adult F1 (B6CBAF1; C57BL/6×CBA/Ca) ovariancells. Colonies of the ESC-like cells were cultured in leukemiainhibitory factor (LIF)-free medium to allow differentiation intoembryoid bodies (EBs). FIG. 9 a, EBs observed on day 4 of culture inLIF-free medium. Immunocytochemistry of EBs was used to detect threegerm layer-specific differentiation using the specific markers of S-100(b; ectodermal), nestin (c; ectodermal), smooth muscle actin (d;mesodermal), Desmin (e; mesodermal), α-fetoprotein (f; endodermal), andTroma-1 (g; endodermal-specific). FIG. 9 b, Real-time PCR analysis ofthe EBs derived from E14 ES cells (h) or the ESC-like cells (i) wasperformed to detect the expression of stem cell-specific or three germlayer-specific genes. The stem cell-specific Oct-4 and Nanog genes orthe three germ layer-specific Ncam (ectoderm), Nestin (ectoderm), Smoothmuscle actin (mesoderm), Desmin (mesoderm), α-fetoprotein (endoderm),and Troma1 (endoderm) genes were used and real-time PCR analysis wasperformed on day 0 (d0; ES or ESC-like cells), day 7 (d7; using EBs),and day 21 (d21; using EBs). Regardless of EB origin, the levels ofOct-4 and Nanog expression decrease with increasing time of EB culture.In contrast, the expression levels of Ncam, Nestin, Smooth muscle actin,Desmin, α-fetoprotein, and Troma1 are higher in EBs maintained for 21days than in either ESC-like cells or EB maintained for 7 days. E14 EScells and their derived EBs (h); ESC-like cells and their derived EBs(i). N/D, not detected. Scale bar=100 μm.

FIG. 10. In vivo differentiation of embryonic stem cell (ESC)-like cellsafter subcutaneous transplantation into NOD-SCID mice. The teratomacontained (a) ectodermal, neuroepithelial rosettes (scale bar=100 μm),(b) ectodermal, keratinized stratified squamous epithelial cells (scalebar=100 μm), (c) mesodermal, osteoid islands showing bonydifferentiation (scale bar=100 μm), (d) mesodermal muscle (scale bar=50μm), (e) endodermal pancreatic tissue (scale bar=50 μm), and (f)endodermal, ciliated columnar epithelial cells (arrows) (scale bar=50μm). This picture depicts the teratoma that resulted from thetransplantation of ScB6CD-SNU-1.

FIG. 11. Production of somatic chimeras by the aggregation of embryonicstem cell (ESC)-like cells derived from the coculturing of F1 (B6D2F1;C57BL/6×DBA2) embryonic fibroblasts and adult F1 (B6CBAF1;C57BL/6×CBA/Ca) ovarian cells with 8-cell embryos of the ICR strain.ESC-like cells (10-15 cells) were aggregated with 8-cell embryos and theblastocysts derived from the aggregated embryos were transplanted intothe uterine horn of surrogate mothers. Live offspring with differentcoat and skin colors are determined to be somatic chimeras. Asterisksand arrows indicate the surrogate mothers and 10-day-old chimericprogenies, respectively.

FIG. 12. Karyotyping of established embryonic stem cell (ESC)-likecells, in vivo-differentiated teratomas, and embryoid bodies (EBs) at 7,14, and 21 days post-differentiation. FACS analysis was used tokaryotype the established ESC-like cells of the ScB6CD-SNU-1 line. (a)ESC-like cells collected from late (30 passages) passages; (b) teratomasretrieved 8 weeks after transplantation into a SCID mouse; and EBs after7 (c), 14 (d) and 21 (e) days of culture in leukemia inhibitory factor(LIF)-free medium. The ESC-like cells are tetraploidy, while there areno tetraploid cells in the teratomas. The number of diploid cellsincreases as spontaneous differentiation in vitro progresses up to 21days.

FIG. 13 schematically represents one specific example of the presentprocess for preparing ESC-like cells the second somatic cell populationcontaining adult stem cells with the help of ovarian niche.

FIG. 14 schematically represents one specific example of the presentprocess for preparing ESC-like cells the first cell populationcontaining adult stem cells.

FIGS. 15 a-15 b show the expression of genes related to pluripotency invarious tissues of adult mice. FIG. 2 a, Expression of Oct-4, Nanog,Rex-1, and Cripto genes in the brain, heart, lung, liver, stomach,kidney, ovary, small intestine, skin, and spleen retrieved from8-week-old, female mice. Oct-4 expression is detected in the ovary,small intestine, and spleen, while all the tissues examined express theNanog gene. Most of the organs, with the exceptions of the stomach andskin express the Cripto gene. FIG. 2 b, Expression of Oct-4, Nanog,Rex-1, Cripto, Dnmt3b, Tert, and Lif Rc genes in individual ovariesretrieved from 8-week-old female mice. E14 embryonic stem cells (ESC)were used as the control cells. All the genes tested are expressed inthe E14 ESCs. +, Reverse transcription using isolated total RNA; −,non-reverse transcription using isolated total RNA.

FIG. 16 represents quantification of pluripotency-specific geneexpression in mouse ovaries retrieved from different animals. Theexpression levels of the Oct-4 (a), Nanog (b), Rex-1 (c), Cripto (d),Tert (e), Lif Rc (f), and Dnmt3b (g) genes in the ovaries retrieved fromdifferent mice were measured by real-time PCR. E14 embryonic stem cellswere used as the positive control cells. Different levels of geneexpression are detected among the ovaries examined. N/D, not detected.

FIG. 17 shows immunostaining results of the cells dissociated from thespleen (a), small intestine (b) and ovary (c) with stem cell-specific,Oct-4 and Nanog. Cells of each organ were dissociated with enzymetreatment and the 1×103 dissociated cells were subsequently stained withanti-Oct-4 (green fluorescence) or anti-Nanog (red fluorescence)antibody. Confocal microscope image show that there are Oct-4-positivecells in splenic and small intestinal cells, while no Nanog-positivecells are found. There are the ovarian cells positively concomitantlystained with Oct-4 and Nanog. Phase-contrast image show that the Oct-4-and Nanog-positive cells (arrows) are mixed with several types of cells.Scale bar=20 μm.

FIG. 18 shows morphology of colony-forming cells derived from thecoculturing of adult ovarian cells and embryonic fibroblasts in mice. a,E14 embryonic stem cells. b, Colony-forming cells on day 7 of primaryculture. Colony-forming cells after ten subpassages (day 37 of culture;c) and after 20 subpassages (day 76 of culture; d). Scale bar=50 μm.

FIG. 19 represents in situ hybridization of adult ovarian tissue for thedetection of Oct-4 and Nanog mRNA expression, and double immunostainingof stem cell-specific Nanog, germ cell-specific Vasa, folliclecell-specific AMH (anti-mullerian hormone) or mesenchymal cell-specificCD44 positive ovarian tissue-dissociated cells with Oct-4. (a) Image ofovarian tissue after in situ hybridization. The ovaries retrieved from8-week-old, F1 hybrid mice were provided for the hybridization usingOct-4- and Nanog-specific mRNA probes. Oct-4 and Nanog mRNA expressionis evident in the ovarian medullae (OM), while the peripheral (thecacell) region of ovarian follicles (OF) with various sizes also shows themRNA expression. (CL) corpus luteum. Scale bar=100 μm. (b) Ovarian cellswere double-immunostained immediately after dissociation. Nanog-, Vasa-or CD44-positive cells are concomitantly immunustained with anti-Oct-4antibody, while AMH-positive cells are not positive for Oct-4.Phase-contrast image of Nanog-, Vasa-, CD44- or AMH-positive cells(arrows) shows that the vasa-positive cells are smaller than othercells. Scale bar=10 μm.

FIG. 20 represents the characterization of colony-forming cells derivedfrom the coculturing of adult ovarian cells and embryonic fibroblasts inmice. a, Characterization using embryonic stem cell (ESC)-specificmarkers. Antibodies against stage-specific embryonic antigen (SSEA)-1,SSEA-3, and SSEA-4, and Oct-4, integrin α6, and integrin β1, as well asalkaline phosphatase (AP) were used for the characterization, and theE14 ESC line was employed as the positive control. Similar to the E14ESCs, the colony-forming cells are positive for SSEA-1, Oct-4, integrinα6, integrin β1, and AP. However, both the E14 ESCs and the establishedcells are negative for SSEA-3 and the SSEA-4. Scale bar=50 μm. b,Pluripotent cell-specific gene expression of the E14 ESCs and thecolony-forming cells was monitored by RT-PCR and similar gene expressionwas detected. Both lines established were characterized, but the imagefrom OSC-B6D2-SNU-1 is depicted on behalf of the established cells.

FIG. 21 represents telomerase activities of colony-forming cellsdetected by the telomeric repeat amplification protocol assay. Two celllines were evaluated. The ladder of telomerase products amplified by PCRwas shown with six-base increments starting at 50 nucleotides at theportion indicated by the asterisk. All of the cell lines express highlevels of telomerase activity. Lane 1, positive control (E14 embryonicstem cells); lane 2, MEFs; lanes 3-4, colony-forming cells ofOSC-B6D2-SNU-1 and OSC-B6D2-SNU-2, respectively; lane 5, PCR controlwithout the addition of template, respectively.

FIG. 22 represents karyotyping and sexing of the establishedcolony-forming cells. G-banding of air-dried chromosomes in theestablished colony-forming cells were undertaken for exact karyotyping(a) and the population of diploid cells were estimated with a flowcytometry (b). In a and b, the image from OSC-B6D2-SNU-1 is depicted onbehalf of the established lines. PCR analysis was further conductedusing the X chromosome-specific Xist and Y chromosome-specific Zfyprimers (c; Lane 1, tail cells with XX; lane 2, neonatal skin fibroblastwith XY; lane 3, established OSC-B6D2-SNU-1; lane 4, OSC-B6D2-SNU-2;lane 5, testis cells). Diploidy is detected in both established linesand both two lines express the X chromosome-specific Xist gene but notthe Zfy gene.

FIG. 23 shows characterization of colony-forming cells by fluorescenceactivated cell sorting (FACS) using mesenchymal stem cell (MSC)-specificmarker CD44 (a) and Sca-1 (b), epithelial stem cell-specific marker CD34(c) and hematopoietic stem cell-specific marker CD45 (d). The greencolor represented peaks of MSCs used as the control. E14 embryonic stemcells and established colony-forming cells are all negative foranti-CD44, anti-Sca-1, anti-CD34 and anti-CD45 antibodies.

FIG. 24 shows immunostaining results of colony-forming cells with thegerm cell-specific markers Vasa and Fragilis, and with the follicularcell-specific marker AMH. Two lines of colony-forming cells wereprovided for the staining and the ovarian cells before seeding and E14embryonic stem cells (ESCs) were provided as the control cells. Thereare the cells positive for Vasa (a), Fragilis (b) or AMH (c) staining inthe ovarian cells, while the colony-forming cells retrieved at thetwentieth subpassage and E14 ESCs are not positive for those markers.Scale bar=50 μm.

FIG. 25 represents spontaneous differentiation of colony-forming cellsin vitro and in vivo. a, In vitro-differentiation of colony-formingcells into embryoid bodies (EBs) by culturing in leukemia inhibitoryfactor free-culture medium. (a1) EBs observed on day 4 of culture.Immunocytochemistry of EBs was undertaken to detect three germlayer-specific differentiation using the specific markers of S-100 (a2;ectodermal), nestin (a3; ectodermal), smooth muscle actin (a4;mesodermal), Desmin (a5; mesodermal), α-fetoprotein (a6; endodermal),and Troma-1 (a7; endodermal-specific). Scale bar=100 μm. b, In vivodifferentiation of colony-forming cells by subcutaneous transplantationinto NOD-SCID mice. The teratoma contained (b1) endodermal, glandularepithelium-Goblet cell like (arrow head), (b2) endodermal, exocrinepancreas, (b3) ectodermal, stratified squamous epithelium (arrow), (b4)ectodermal, neuroepithelial rossettes, (b5) mesodermal, skeletal musclebundles, and (b6) mesodermal, bone tissue (arrow). Scale bar=50 μm. Bothlines established were characterized, but the image from OSC-B6D2-SNU-1is depicted on behalf of the established cells.

FIG. 26 represents neuronal cell differentiation of colony-forming cellsderived from the coculturing of adult ovarian cells and embryonicfibroblasts in mice. a, Nestin-positive and b, Tuj1-positive neuronsgenerated 14 days after replating on fibronectin. c, O4-positiveoligodendrocyte generated 8 days and (d) GFAP-positive astrocytesgenerated 15 days after the replating. e-h, Phase contrast images ofdifferentiated colony-forming cells into nestin-positive neurons,O4-positive oligodendrocyte and GFAP-positive astrocyte in modifiedN2B27 medium, respectively. Both lines established were provided forneuronal cell differentiation, but the image from OSC-B6D2-SNU-1 isdepicted on behalf of the established cells (Scale bar=10 μm).

DETAILED DESCRIPTION OF THIS INVENTION

In one aspect of this invention, there is provided a method forpreparing an embryonic stem cell (ESC)-like cell, which comprises thesteps of: (a) obtaining a first cell population from a mammalian tissueor body fluid, wherein the first cell population comprises adult stemcells; (b) obtaining a second somatic cell population from a mammaliantissue, wherein the mammalian tissue is different from the mammaliantissue in step (a) and the second cell population is different from thefirst cell population; (c) coculturing the first cell population and thesecond cell population in a medium for a period of time sufficient toform a colony from either the first cell population or the second cellpopulation; and (d) subculturing a cell from the colony in a medium fora period time sufficient to prepare the ESC-like cell.

The present invention is directed to a novel approach for establishingembryonic stem cell (ESC)-like cells having pluripotency from mammaliantissue cells without undertaking SCNT and without using embryos and evengametes. Therefore, the present method is also expressed as methods forpreparing ESC-like cells without using embryo. The present inventionmakes it possible and practicable to establish an immune reaction-free,patient-specific cell therapy without human cloning. To our bestknowledge, the present invention provides for the first time thegeneration of ESC-like cells from mammalian cells, e.g., differentiatedsomatic cells, with no help of gamete manipulation. According to thepresent method, two cell populations can be induced to form ESC-likecells by adjusting culture conditions and/or environment.

The term “embryonic stem cell (ESC)-like cell” is used herein to referto a cell having pluripotency that is induced by the present method, toexhibit a property of an embryonic stem cell, including, but not limitedto, proliferation without transformation, continuous proliferation,self-renewal and capacity of developing into any cell derived from thethree main germ cell layers, and the like. That is, the ESC-like cellsinduced by the present invention acquire an embryonic-like stage.

The present invention will be described in more detail as follows:

Preparation of First Cell Population Containing Adult Stem Cells

The first cell population containing adult stem cells may be obtained byvarious conventional methods from a multitude of sources. The source forthe first cell population containing adult stem cells may comprise anymammalian tissue or fluid previously known to contain adult stem cells.

The term “adult stem cell” used herein refers to an undifferentiated(unspecialized) cell that occurs in a differentiated (specialized)tissue, renews itself, and becomes specialized to yield all of thespecialized cell types of the tissue from which it originated.

According to a preferred embodiment, the mammalian tissue or body fluidas sources for the adult stem cells-containing first cell population isderived from ovary, testis, bone marrow, peripheral blood, umbilicalcord blood, amniotic fluid, brain, blood vessel, skeletal muscle,epithelia of skin or gastrointestinal tract, cornea, dental pulp oftooth, retina, liver, spleen or pancreas. For example, stem cellsderived from brain have been isolated in the subventricular zone,ventricular zone and hippocampus of the CNS (central nervous system).Bone marrow has been reported to have hematopoietic stem cells andmesenchymal (stromal) stem cells.

More preferably, the mammalian tissue or body fluid as sources for theadult stem cells-containing first cell population is ovary, liver,stomach, skin or spleen, most preferably, ovary.

The first cell population containing adult stem cells for preparingESC-like cells may be obtained by various methods known in the art. Forexample, cells may be obtained by disassociation of tissue by mechanical(e.g., chopping and mincing) or enzymatic means (e.g., trypsinization)to obtain a cell suspension followed by culturing the cells until aconfluent monolayer is obtained. The cells may then be harvested andprepared for cryopreservation. The isolation of somatic cells, forexample, ovarian cell, is described in Examples.

The first cell population containing adult stem cells may furthercontain any type of cells that contains a genome or genetic material(e.g., nucleic acid), such as a somatic cell and germ cell. The term“somatic cell” as used herein refers to a differentiated cell. The cellmay be a somatic cell or a cell that is committed to a somatic celllineage. Preferably, the cell useful in this invention is adifferentiated somatic cell.

The first cell population may be obtained from tissues at anydevelopment stage, e.g., post-puberty mammalian tissue, pre-embryonic,embryonic, neonatal or fetal tissue at any time after fertilization.According to a preferred embodiment, the first cell population isobtained from a mammalian tissue or body fluid at any development stageafter birth, more preferably, from a post-puberty mammalian tissue orbody fluid. The term “post-puberty mammalian” is intended to refer to amammal after a puberty stage in development of mammals. That is, theterm “post-puberty mammalian” has the identical meaning to the term“adult mammalian” generally used in this art. There is no intendeddistinction between the terms “post-puberty mammalian” and “adultmammalian”, and these terms will be used interchangeably. For example,the average age at puberty for mice, cow, pig and human isapproximately, 3 to 4 weeks, 10 to 14 months, 2 to 3 months and 10 to 15years, respectively.

Where cells from the ovary, i.e., ovarian cells are used, it ispreferred that they are substantially freed from ovarian stromal cells,oocytes, and preantral and antral follicles. The removal of oocytes, andpreantral and antral follicles from ovarian cell preparations may becarried out using a 40-μm cell strainer. Ovarian cells containing adultstem cells used in this invention may contain adult somatic cells,mesenchymal stem cells, primordial follicle and/or ovarian stem cells.

The first cell population provides the second cell population withmicroenvironment (a niche) suitable in the transformation(dedifferentiation) of cells into ESC-like cells. Without wishing to bebound by theory, it is believed that the niche may secrete and/orprovide the factors required to induce dedifferentiation. The term usedherein “niche” refers to components (cells and/or substances) composedof tissues or organs (e.g., ovary) supporting the development andproliferation of tissue cells such as stem cells and other somaticcells. In the cases of using ovarian cells, the ovarian niche isprovided.

In contrast, under suitable conditions and environment, the first cellpopulation itself is dedifferentiated to ESC-like cells, rather thanproviding a niche for the dedifferentiation of the second cellpopulation.

According to a preferred embodiment, the first cell population is aheterogeneous cell population comprising at least two cell types. Forexample, where the first cell population is derived from the ovary, itmay comprise mesenchymal stem cells as well as adult somatic cells,primordial follicle and/or ovarian stem cells.

According to a preferred embodiment, the first cell population used inthis invention is originated from human, bovine, sheep, ovine, pig,horse, rabbit, goat, mouse, hamster or rat, more preferably, human,mouse or rat.

The genome of the first cell population used in this invention may bethe naturally occurring genome, for example, or the genome may begenetically altered to comprise a transgenic sequence. Preferably, thefirst cell population used in this invention has a naturally occurringgenome, especially, for cell transplantation therapy.

Preparation of Second Somatic Cell Population

It is necessary in the present invention that the type of the secondsomatic cell population is different from that of the first cellpopulation.

The second somatic cell population may be obtained by various methodsknown in the art. For example, cells may be obtained by disassociationof tissue by mechanical (e.g., chopping and mincing) or enzymatic means(e.g., trypsinization) to obtain a cell suspension followed by culturingthe cells until a confluent monolayer is obtained. The cells may then beharvested and prepared for cryopreservation. The isolation of somaticcells, for example, fibroblasts, is described in Examples.

The cells may be any type of somatic cells that contains a genome orgenetic material (e.g., nucleic acid). The term “somatic cell” as usedherein refers to a differentiated cell. The cell may be a somatic cellor a cell that is committed to a somatic cell lineage. Preferably, thecell useful in this invention is a differentiated somatic cell. Thesomatic cell may be originated from a mammalian animal or from a celland/or tissue culture system. If taken from a mammalian animal, theanimal may be at any stage of development, for example, an embryo, afetus or an adult. Additionally, the present invention may utilizeembryonic somatic cells.

Suitable somatic cells include fibroblasts (e.g., primary fibroblasts),epithelial cells, muscle cells, cumulous cells, neural cells, andmammary cells. Other suitable cells include hepatocytes and pancreaticislets. Preferably, the second cell population includes fibroblasts andepithelial cells, most preferably fibroblasts.

According to a preferred embodiment, the cell used in this invention isoriginated from human, bovine, sheep, ovine, pig, horse, rabbit, goat,mouse, hamster or rat, more preferably, human, mouse or rat.

The genome of the somatic cell used in this invention may be thenaturally occurring genome, for example, or the genome may begenetically altered to comprise a transgenic sequence. Preferably, thesomatic cell used in this invention has a naturally occurring genome,especially, for cell transplantation therapy. It is preferable that thecell used in this invention is a somatic cell having a diploidkaryotype.

According to a preferred embodiment, mitotically inactive cells are usedas a cell source for the second cell population. Mitotically inactivecells may be readily prepared for arresting cell cycle by variousmethods known to one of skill in the art. For example, mitoticallyinactive cells may be produced by exposure to gamma radiation (e.g.,4000 Rads of gamma radiation) or treatment with mitomycin C.

According to a preferred embodiment, the second somatic cell populationhas adherent characteristics to culture plates. Such adherent potentialpermits the second somatic cell population to support the growth of thefirst cell population. According to a preferred embodiment, the secondsomatic cell population is a homogenous population substantiallycomprising one somatic cell type.

The second somatic cell population is dedifferentiated to ESC-like cellswith help of the first cell population.

In contrast, under suitable conditions and environment, the secondsomatic cell population provides environments for the dedifferentiationof the first cell population, rather than dedifferentiating itself toESC-like cells. In such case, the second somatic cell population mayserve as feeder cells.

Coculturing for Forming Colonies Comprising Esc-Like Cells

Two types of cells prepared above, especially, the first and second cellpopulations, are then cocultured in a medium for a period of timesufficient to form colonies comprising ESC-Like cells havingpluripotency.

Preferably, the medium comprises a factor for inhibiting the celldifferentiation. The differentiation inhibitory factor includes leukemiainhibitory factor and nm23-H2/nucleoside diphosphate kinase (NDPK)-B.Most preferably, the medium for coculturing comprises leukemiainhibitory factor.

Without wishing to be bound by theory, it is believed that the firstcell population or the second somatic cell population and leukemiainhibitory factor, especially, a high dose of leukemia inhibitory factorprovide the cells with microenvironment (or niche) suitable in thetransformation (dedifferentiation) of cells into pluripotent ESC-likecells. Without wishing to be bound by theory, it is believed that themicroenvironment (or niche) may trigger cell-to cell interaction andsecrete the factors for acquiring pluripotency.

According to a preferred embodiment, the coculturing step is carried outin the presence of higher concentrations of leukemia inhibitory factor(LIF). The term “higher concentration” used herein with reference to LIFmeans at least 3,000 units/ml, preferably at least 4,000 units/ml, morepreferably at least 5,000 units/ml, still more preferably, 4,000-6000units/ml of LIF, most preferably, about 5000 units/ml of LIF. Accordingto a more preferred embodiment, the culturing in step (c) is carried outin the presence of at least 3,000 units/ml of LIF, still more preferablyat least 4,000 units/ml, still yet more preferably at least 4,000-6,000units/ml, and most preferably about 5,000 units/ml of LIF. Therelatively high concentration of LIF is very advantageous for theproduction of pluripotent stem cells with higher yield.

A medium useful in this step includes any conventional medium forobtaining mammalian ES cells known in the art. For example, the mediumincludes Dulbecco's modified Eagle's medium (DMEM), knock DMEM, DMEMcontaining fetal bovine serum (FBS), DMEM containing serum replacement,Chatot, Ziomek and Bavister (CZB) medium, Ham's F-10 containing fetalcalf serum (FCS), Tyrodes-albumin-lactate-pyruvate (TALP), Dulbecco'sphosphate buffered saline (PBS), and Eagle's and Whitten's media.Preferably, the culture medium is DMEM containing LIF (leukemiainhibitory factor) supplemented with β-mercaptoethanol, nonessentialamino acids, L-glutamine, antibiotics (preferably, penicillin andstreptomycin) and/or FBS (fetal bovine serum). The detailed descriptionof media is found in R. Ian Freshney, Culture of Animal Cells, A Manualof Basic Technique, Alan R. Liss, Inc., New York, WO 97/47734 and WO98/30679, the teachings of which are incorporated herein by reference intheir entities.

The period of time for forming colonies is not particularly restricted,preferably in the range of 4-9 days, more preferably 6-8 days, mostpreferably about 7 days.

The colony can be derived from either the first cell population or thesecond somatic cell population. Without wishing to be bound by theory,the determination is based on the genetic background, environmentalsusceptibility and/or type of cells.

As exemplified in Examples, cells to be dedifferentiated are determineddepending on genetic background of cells. Specifically, where ovariancells as the first cell population are obtained from B6CBAF1 mice(produced by mating C57BL/6 mice with CBA/Ca mice) and fibroblasts asthe second somatic cell population are obtained from B6D2F1 mice(produced by mating C57BL/6 mice with DBA2 mice), the second somaticcell population is dedifferentiated to ESC-like cells. Unlikely, whereovarian cells as the first cell population are obtained from B6D2F1 miceand fibroblasts as the second somatic cell population are obtained fromICR mice, the first cell population is dedifferentiated to ESC-likecells. These findings demonstrate that the genetic background of cellsused in coculturing with two cell types determines cells to bededifferentiated to ESC-like cells. In Examples, cells originated fromB6D2F1 mice are dedifferentiated to ESC-like cells.

The present inventors also examined characteristics of cells from B6D2F1mice and found that stress-defense genes (e.g., Hspa9a, Bmi1, Hspa1b,Pdha2 and Txrnd3) are expressed in a relatively low level and reactiveoxygen species are generated in a relatively high level (data notshown). That is, cells originated B6D2F1 mice exhibit high environmentalsusceptibility. On the basis of these analysis results, it could berecognized that the environmental susceptibility of cells used incoculturing with two cell types determines cells to be dedifferentiatedinto ESC-like cells. Among two type cells in the coculturing system,cells having higher environmental susceptibility are very likely to bededifferentiated into ESC-like cells.

Subculturing Cells Forming Colonies to Prepare ESC-Like Cells

For preparing ESC-like cells, colony-forming cells are subcultured in amedium for a period of time sufficient to prepare the ESC-like cell.

According to a preferred embodiment, the subculturing step is carriedout in the presence of relatively low concentrations of leukemiainhibitory factor (LIF). More preferably, the subculture is carried outin the presence of no more than 2,000 units/ml of LIF, still morepreferably 800-1,200 units/ml, most preferably 1,000 units/ml of LIF.

Detailed descriptions of medium and feeder cell layers for thesubculturing step follow those for the culturing in step (c) discussedhereinabove.

ESC-like cells prepared by this invention may be maintained for morethan 3 months with 25 passages. Therefore, where the subculturing isperformed without substantially undergoing changes (e.g., genetically orbiologically) for a longer period of time, the present invention is alsoexpressed as a method for preparing an ESC-like cell line havingpluripotency.

According to a preferred embodiment, where the ESC-like cell is preparedfrom the colony derived from the second cell population, the ESC-likecell has a tetraploid karyotype. When the ESC-like cell havingtetraploid karyotype is induced to differentiate, the ESC-like cellbecomes to have a diploid karyotype. In accordance with this invention,diploid-to-tetraploid and tetraploid-to-diploid shifts occur during theacquisition of sternness by reprogramming and dedifferentiation andduring differentiation into somatic cells, respectively. These novelfindings form mechanism and theory underlying this invention.

In contrast to this, where the ESC-like cell is prepared from the colonyderived from the first cell population, the ESC-like cell has a diploidkaryotype.

The preparation of pluripotent ESC-like cells may be evaluated by makerassays using alkaline phosphatase (AP), anti-stage-specific embryonicantigen (SSEA) antibodies such as anti-SSEA-1, anti-SSEA-3 andanti-SSEA-4 antibodies, anti-integrin α6 antibody, and anti-integrin β1antibody. In addition, the pluripotent stem cells finally prepared bythe invention may be confirmed by analyzing their potentials to formembryonic body in the absence of LIF and teratoma. Meanwhile, thekaryotyping and DNA microsatellite analysis of ESC-like cells finallyproduced may reveal that they are originated from tissue cells (e.g.,ovarian cells or fibroblasts) targeted to be dedifferentiated.

According to a preferred embodiment, ESC-like cells originated fromeither the first cell population or the second somatic cell populationshow a positive reactivity to alkaline phosphatase, and to an antibodyagainst each of alkaline phosphatase, stage specific embryonic antigen(SSEA)-1, integrin α6, integrin β1 and Oct-4, and a negative reactivityto an antibody against each SSEA-3 and SSEA-4.

According to a preferred embodiment, pluripotent ESC-like cells producedby the present method express at least one stem-cell specific geneselected from the group consisting of Oct-4, Nanog, Rex-1, Cripto,Dnmt3b, Tert, Lif Rc, Stat3, Bmp4, Fgf4, Foxd3, Sox2, CD9, and Gdf3.According to a preferred embodiment, ESC-like cells produced by thepresent method show a negative reactivity to an antibody againsttissue-specific stem cell markers (Sca-1 and CD44 for mesenchymal stemcells, CD34 for epithelial stem cells, CD45 for hematopoietic stem cell,and Fragilis and Vasa for germline stem cells). Preferably, ESC-likecells established by the present method show no reactivity to anantibody against follicle cell-specific markers [e.g., AMH(anti-mullerian hormone)].

As discussed hereinabove, the present method provides two approaches forpreparing ESC-like cells by use of mammalian tissue cells.

The first approach uses a niche derived from mammalian tissues or bodyfluids. Specifically, the first approach comprises the steps of: (a)obtaining an ovarian niche from a mammalian ovary wherein the ovarianniche comprises adult stem cells; (b) obtaining a somatic cellpopulation from a mammalian tissue to be dedifferentiated, wherein themammalian tissue is different from the ovarian niche in step (a) and thesomatic cell population is different from the ovarian niche; (c)coculturing the ovarian niche and the somatic cell population in amedium for a period of time sufficient to form a colony from the somaticcell population; and (d) subculturing a cell from the colony in a mediumfor a period time sufficient to prepare the ESC-like cell.

In particular, the second approach comprises the steps of: (a) obtaininga first cell population from a mammalian tissue or body fluid, whereinthe first cell population comprises adult stem cells; (b) obtaining asecond somatic cell population from a mammalian tissue, wherein themammalian tissue is different from the mammalian tissue in step (a) andthe second cell population is different from the first cell population;(c) coculturing the first cell population and the second cell populationin a medium containing leukemia inhibitory factor for a period of timesufficient to form a colony from the first cell population; and (d)subculturing a cell from the colony in a medium containing leukemiainhibitory factor for a period time sufficient to prepare the ESC-likecell.

The detailed descriptions of the first and second approaches followthose of the present method for preparing ESC-like cells discussedhereinabove.

In another aspect of this invention, there is provided a mammaliantissue-derived embryonic stem cell-like cell, wherein the embryonic stemcell-like cell has pluripotency and is prepared by culturing cells froma post-puberty mammalian tissue; and the embryonic stem cell-like cellis not prepared by a somatic cell nuclear transfer.

In still another aspect of this invention, there is provided a mammaliantissue or body fluid-derived embryonic stem cell (ESC)-like cell,wherein the ESC-like cell has pluripotency; the ESC-like cell isprepared by coculturing (i) an adult stem cell-containing first cellpopulation from a mammalian tissue or body fluid and (ii) a secondsomatic cell population from a mammalian tissue different from themammalian tissue of (i); and the ESC-like cell is not prepared by asomatic cell nuclear transfer.

In further aspect of this invention, there is provided a mammaliantissue or body fluid-derived embryonic stem cell (ESC)-like cell line,wherein the ESC-like cell has pluripotency; the ESC-like cell isprepared by coculturing (i) an adult stem cell-containing first cellpopulation from a mammalian tissue or body fluid and (ii) a secondsomatic cell population from a mammalian tissue different from themammalian tissue of (i); and the ESC-like cell is not prepared by asomatic cell nuclear transfer.

The mammalian tissue-derived pluripotent ESC-like cell of this inventionis firstly presented without undertaking human cloning and SCNT. Wherethe somatic cell is derived from patient oneself, the pluripotentESC-like cell enables to provide an immune reaction-free,patient-specific cell therapy without undertaking human cloning andSCNT.

The ESC-like cell of this invention is pluripotent. The term“pluripotent” means that cells have the ability to develop into any cellderived from the three main germ cell layers. When transferred into SCIDmice, a successful somatic cell-derived pluripotent stem cell willdifferentiate into cells derived from all three embryonic germ layers.In addition, when cultured in the absence of LIF, the somaticcell-derived pluripotent stem cell of this invention forms an embryonicbody being positive for markers specific for any of the three germlayers: neural cadherin adhesion molecule and S-100 for the ectodermallayer; muscle actin and desmin for the mesodermal layer; andα-fetoprotein and Troma-1 for endodermal cells.

The term “ESC-like cell line” used herein means a culture of cellsobtained by long-term culture of the pluripotent ESC-like cells. TheESC-like stem cell line is stabile in the senses that it can be culturedfor a period of time without substantially undergoing changes (e.g.,genetically or biologically).

According to a preferred embodiment, the mammalian tissue or body fluidfor the adult stem cell-containing first cell population is derived fromovary, bone marrow, peripheral blood, umbilical cord blood, amnioticfluid, brain, blood vessel, skeletal muscle, epithelia of skin orgastrointestinal tract, cornea, dental pulp of tooth, retina, liver,spleen or pancreas. Most preferably, the mammalian tissue is derivedfrom ovary.

According to a preferred embodiment, the first cell population is aheterogeneous population comprising at least two cell types. Preferably,the second somatic cell population is a homogenous populationsubstantially comprising one somatic cell type. The somatic cell,preferably, is fibroblast or epithelia cell. It is preferred that thesecond somatic cell population is a mitotically inactive cell. Inaddition, it is preferred that the second somatic cell population hasadherent characteristics to culture plates.

According to a preferred embodiment, the first cell population isobtained from a post-puberty mammalian tissue or body fluid.

It is preferable that the first cell population comprising stem cells isan ovarian cell, more preferably, substantially freed from ovarianstromal cells. It is more preferable that the ovarian cell issubstantially freed from oocytes and preantral and antral follicles.

The ESC-like cells of this invention may be originated from variousanimals, preferably, human, bovine, sheep, ovine, pig, horse, rabbit,goat, mouse, hamster or rat.

According to a preferred embodiment, when the ESC-like cell isoriginated from the second cell population, the ESC-like cell has atetraploid karyotype. Where the ESC-like cell with tetraploid karyotypeis induced to differentiate, the ESC-like cell has a diploid karyotype.

Unlikely, when the ESC-like cell is originated from the first cellpopulation, the ESC-like cell has a diploid karyotype.

The mammalian tissue cell-derived ESC-like cell has the same genotype asits progenitor cell. In addition, ESC-like cell of this inventionexhibits some characteristics common to embryonic stem cells, forexample, being stainable with alkaline phosphatase (AP) and capable offorming an embryonic body and teratoma.

The term “stainable” used herein with reference to embryonic stem cellsmeans that cells are positively stained with or reactive to cell surfacebinding ligands such as AP, anti-SSEA antibody, anti-integrin α6antibody and anti-integrin β1 antibody.

According to a preferred embodiment, when the ESC-like cell isoriginated from the first cell population, the ESC-like cell shows apositive reactivity to alkaline phosphatase, and to an antibody againsteach of stage specific embryonic antigen (SSEA)-1, integrin α6, integrinβ1 and Oct-4, and a negative reactivity to an antibody against eachSSEA-3 and SSEA-4. Preferably, the ESC-like cell expresses at least onestem-cell specific gene selected from the group consisting of Oct-4,Nanog, Rex-1, Cripto, Dnmt3b, Tert, Lif Rc, Stat3, Bmp4, Fgf4, Foxd3,Sox2, CD9, and Gdf3. More preferably, the ESC-like cell shows a negativereactivity to an antibody against Sca-1, CD44, CD34, CD45, Fragilis,Vasa and/or AMH (anti-mullerian hormone).

Exemplarily, the ESC-like cell originated from the first cell populationis OSC-B6D2-SNU-1 under accession No. KCLRF-BP-00148.

According to a preferred embodiment, when the ESC-like cell isoriginated from the second cell population, the ESC-like cell shows apositive reactivity to alkaline phosphatase, and to an antibody againsteach of stage specific embryonic antigen (SSEA)-1, integrin α6, integrinβ1 and Oct-4, and a negative reactivity to an antibody against eachSSEA-3 and SSEA-4.

The exemplified ESC-like cell originated from the second cell populationis tScB6CD-SNU-1 under accession No. KCLRF-BP-00135.

It is well known that ES cells or ESC-like cells are capable ofdifferentiating into any type of cells. Therefore, the ESC-like of thisinvention may be a good source providing various types of cells. Forexample, the ESC-like cell may be induced to differentiate intohematopoietic cells, nerve cells, beta cells, muscle cells, liver cells,cartilage cells, epithelial cell, urinary tract cell and the like, byculturing it a medium under conditions for cell differentiation. Mediumand methods which result in the differentiation of ES cells are known inthe art as are suitable culturing conditions (Palacios, et al., PNAS.USA, 92:7530-7537 (1995); Pedersen, J. Reprod. Fertil. Dev., 6:543-552(1994); and Bain et al., Dev. Biol, 168:342-357 (1995)).

The ESC-like cell of this invention has numerous therapeuticapplications through transplantation therapies. The ESC-like cell ofthis invention has application in the treatment of numerous diseases ordisorders such as diabetes, Parkinson's disease, Alzheimer's disease,cancer, spinal cord injuries, multiple sclerosis, amyotrophic lateralsclerosis, muscular dystrophy, diabetes, liver diseases, i.e.,hypercholesterolemia,, heart diseases, cartilage replacement, burns,foot ulcers, gastrointestinal diseases, vascular diseases, kidneydisease, urinary tract disease, and aging related diseases andconditions.

In still further aspect of this invention, there is provided a culturemedium for a culture medium for dedifferentiating a mammalian cellhaving no pluripotency into an embryonic stem cell (ESC)-like cellhaving pluripotency, which comprises a cell population from a mammaliantissue or body fluid, wherein the cell population comprises adult stemcells.

The culture medium of this invention is also described using the term“culture system” and these two terms will be used interchangeably.

Since the culture medium or culture system of this invention is used inthe method of this invention for producing ESC-like cells describedhereinabove, the common descriptions between them will be omitted inorder to avoid undue redundancy leading to the complexity of thisspecification.

According to a preferred embodiment, the mammalian tissue or body fluidis derived from ovary, bone marrow, peripheral blood, umbilical cordblood, amniotic fluid, brain, blood vessel, skeletal muscle, epitheliaof skin or gastrointestinal tract, cornea, dental pulp of tooth, retina,liver, spleen or pancreas. More preferably, the cell populationcomprises ovarian cells. It is preferable that the cell population is aheterogeneous population comprising at least two cell types.

According to a preferred embodiment, the medium further comprises atleast 3,000 units/ml of leukemia inhibitory factor, more preferably, atleast 4,000 units/ml of leukemia inhibitory factor, and most preferably,4,000-6,000 units/ml of leukemia inhibitory factor.

According to a preferred embodiment, the mammalian cell is a somaticcell, more preferably, mitotically inactive somatic cell.

In another aspect of this invention, there is provided a culture mediumfor producing an embryonic stem cell (ESC)-like cell from a mammaliantissue cell, which comprises at least 3,000 units/ml of leukemiainhibitory factor.

According to a preferred embodiment, the medium comprises at least 4,000units/ml of leukemia inhibitory factor, more preferably, 4,000-6,000units/ml of leukemia inhibitory factor, most preferably, about 5,000units/ml of leukemia inhibitory factor.

According to a preferred embodiment, the mammalian tissue cell is a cellobtained from a post-puberty mammalian.

It is preferable that the mammalian tissue cell is derived from ovary,bone marrow, peripheral blood, umbilical cord blood, amniotic fluid,brain, blood vessel, skeletal muscle, epithelia of skin orgastrointestinal tract, cornea, dental pulp of tooth, retina, liver,spleen or pancreas, most preferably, ovary.

According to a preferred embodiment, the mammalian tissue cell is asomatic cell.

According to a preferred embodiment, the medium further comprises amitotically inactive cell layer such as mitotically inactive feederlayer.

The culture medium or system of this invention may further comprise anyingredient contained in conventional media for obtaining mammalian EScells known in the art. For example, the medium may further compriseinorganic salts (e.g., CaCl₂, Fe(NO₃)₃, MgSO₄, NaCl, NaHCO₃ andNaH₂PO₄), energy source (e.g., glucose), buffers, amino acids and/orvitamins (e.g., D-Ca pantothenate, choline chloride, folic acid,nicotinamide), preferably supplemented with β-mercaptoethanol,nonessential amino acids, L-glutamine, antibiotics (preferably,penicillin and streptomycin) and/or FBS (fetal bovine serum). Thecomposition of the culture medium of this invention may prepared inaccordance with that of conventional media such as Dulbecco's modifiedEagle's medium (DMEM), knock DMEM, DMEM containing fetal bovine serum(FBS), DMEM containing serum replacement, Chatot, Ziomek and Bavister(CZB) medium, Ham's F-10 containing fetal calf serum (FCS),Tyrodes-albumin-lactate-pyruvate (TALP), Dulbecco's phosphate bufferedsaline (PBS), and Eagle's and Whitten's media.

The present invention clearly demonstrates that ESC-like cells havingpluripotency can be derived from any cell type, particularly, adultsomatic cell, under microenvironment (niche). In other words, themicroenvironment (niche) allows providing a novel approach to produceESC-like cells. To our knowledge, this is the first invention onestablishing autologous ESC-like cells without using somatic-cellnuclear transfer and gamete manipulation. This approach avoids thesacrifice both of ovulated oocytes having developmental competence andof viable embryos.

The present invention can suggests a new strategy for establishingpluripotent cells from human tissues without undertaking SCNT. Thiswould bypass the ethical issues related to cell/tissue therapy, asimmune-specific pluripotent cells can be derived from any somatic cellof individuals.

The present invention will now be described in further detail byexamples. It would be obvious to those skilled in the art that theseexamples are intended to be more concretely illustrative and the scopeof the present invention as set forth in the appended claims is notlimited to or by the examples.

EXAMPLES For ESC-Like Cells Derived from Fibroblast Materials andMethods Animals

Animals provided for this study were bred at the Laboratory ofEmbryology and Stem Cell Biology, Seoul National University, Korea. Theywere maintained under controlled conditions of lighting (14 L:10 D),temperature (20-22° C.), and humidity (40-60%). Female F1 hybrid(B6CBAF1) mice were produced by mating female C57BL/6 mice with maleCBA/Ca mice and 8-week-old female mice were used as the donors of theovaries. Pregnant C57BL/6 females mated with DBA2 male or outbred (ICR)13.5 days post-coitus (dpc) were euthanized for the derivation of MEFsfor primary culture and subculture. All of the procedures for animalmanagement, breeding, and surgery followed the standard operationprotocols of Seoul National University, and the review board ofExperimental Animal Resources, Seoul National University, approved theusage of animals and the relevant experimental procedures (approval no.SNU-050331-2). Experimental samples were appropriately managed, andquality control of the laboratory facility and equipment was conducted.

In Situ Hybridization

An in situ hybridization detection system kit (DF132-60K; BioGenex, SanRamon, Calif.) and probe (Biognostik, Gottingen, Germany) were usedaccording to the supplied protocol, which was optimized to minimizebackground signals. The collected ovarian tissues were frozen in optimalcutting temperature compound (Tissue-Tek OCT compound; Sakura, Torrance,Calif.) in liquid N₂. The cryo-samples were cut at 10-μm thickness andfixed at room temperature for 5 min in PBS that contained 4% (v/v)formaldehyde (Sigma-Aldrich). The slides were dehydrated in an ethanolseries (70%, 80%, 90%, 95%, and 100%). The air-dried samples wereprehybridized in HybriBuffer-ISH at 30° C. for 3 h. Hybridization wasconducted in HybriBuffer-ISH that contained the Oct-4 or NanogHybriProbe at 40° C. overnight. The slides were washed in 1× sodiumchloride/sodium citrate solution (SSC) at room temperature for 5 min, in0.1×SSC at 45° C. for 15 min, and in 1×PBS supplemented with 0.1% (v/v)Tween-20 (USB, Cleveland, Ohio) at room temperature for 3 min. Theslides were incubated in Power Block Reagent at room temperature for 10min and with the biotinylated anti-fluorescein antibody at roomtemperature for 40 min. After washing with 1×PBS supplemented with 0.1%(v/v) Tween-20, the slides were incubated in streptavidin-alkalinephosphatase conjugate at room temperature for 20 min, and washed with1×PBS supplemented with 0.1% (v/v) Tween-20. Alkaline phosphatase wasactivated by incubating with Activation Buffer at room temperature for 1min. The slides were developed with NBT/BCIP at room temperature for 15min, mounted with Supermount, and observed under a phase-contrastmicroscope (BX51TF; Olympus).

Preparation of MEFs

Embryonic fibroblasts were collected from the 13.5-dpc fetuses (B6D2F1for primary culture and ICR for subculture) of pregnant females. Thevisceral organs, head, and extremities of the fetuses were removed andthe remaining tissue was cut into small pieces. The tissue pieces wereincubated in 0.04% (v/v) trypsin-EDTA (Gibco Invitrogen) for 6 min withagitation, and subsequently centrifuged at 50×g for 2 min. Thesupernatants were diluted in 10% (v/v) FBS-containing DMEM medium (GibcoInvitrogen) and centrifuged at 100×g for 4 min. The pellets of thecollected fibroblasts were suspended and replaced in DMEM medium formonolayer formation. When the fibroblasts formed a confluent monolayer,they were frozen in 10% dimethylsulfoxide (Gibco Invitrogen).

Ovarian Cell Preparation

The ovaries were collected and, after the removal of adherent tissue,the retrieved tissue was chopped using a surgical blade. The specimenswere incubated initially for 30 min in a dissociation medium thatconsisted of a 50:50 (v:v) mixture of 0.25% (v/w) trypsin-EDTA (GibcoInvitrogen) and DMEM (Gibco Invitrogen) that was supplemented with 750U/ml collagenase type I (Sigma-Aldrich, St. Louis, Mo.) and 0.03% (v/v)fetal bovine serum (FBS; HyClone), at 37° C. The dissociated cells werefiltered through a 40-μm cell strainer (BD Falcon, Franklin Lakes, N.J.)and centrifuged at 390×g for 4 min. They were initially seeded into 60mm×10 mm culture dishes. The stromal cells mixed in with the dissociatedcells were removed 30 min after initial seeding and the buoyant cellspresent above the bottomed stromal cells were re-seeded onto MEFmonolayers in the dishes. In some replications, the filtered cells weredirectly seeded onto MEF monolayer without stromal cell removal. Theculture medium was DMEM medium that was supplemented with 0.1 mMβ-mercaptoethanol (Gibco Invitrogen), 1% (v/v) nonessential amino acids(Gibco Invitrogen), 2 mM L-glutamine (Sigma-Aldrich), 1% (v/v)lyophilized mixture of penicillin and streptomycin (Gibco Invitrogen),5,000 U/ml mouse LIF (Chemicon, Temecula, Calif.), and 15% (v/v) FBS(HyClone).

Coculture of MEFs and Ovarian Cells for Establishing ESCs

F1 hybrid (B6D2F1; C57BL/6×DBA2) MEF monolayers were treated with 10μg/ml mitomycin C (Chemicon, Temecula, Calif.) for 3 hrs ingelatin-coated 35-mm tissue culture dishes and subsequently used forestablishing colony-forming cells. The ovarian cells prepared wereadditionally seeded into the dishes that contained MEF monolayers andcultured at 37° C. under 5% CO₂ in a humidified air atmosphere. On day 4of culture, the mixed population of the embryonic fibroblasts andovarian cells reached confluency and subsequently replated on a newmonolayer in 60-mm tissue culture dishes containing culture medium. TheLIF concentration added to the culture medium ranged from 1,000 to 5,000U/ml in primary culture and was fixed at 2,000 U/ml for the subcultures.At the end of the primary culture (on day 7 of culture), colony-formingcells were mechanically removed with a capillary pipette, dissociatedwith 0.25% (v/w) trypsin-EDTA (Gibco Invitrogen) and subcultured withthe ICR MEF monolayer. Subpassaging was conducted in the presence of2,000 U/ml LIF at intervals of 3 days, whereas the medium was changeddaily.

Marker Staining of ESC-Like Cells

For characterization using stem cell-specific markers, colony-formingcells collected at the 20^(th) subpassage were fixed in 4% (v/v)formaldehyde (Sigma-Aldrich) at room temperature for 10 min. Thereactivity of the colony-forming cells to alkaline phosphatase wasassessed with Fast Red TR/naphthol AS-MX phosphate (Sigma-Aldrich).Monoclonal antibodies against stage-specific embryonic antigens (SSEA)-1(MC-480), SSEA-3 (MC-631), SSEA-4 (MC-813-70), integrin α6 (P2C62C4),and integrin β1 (MH-25) were supplied by the Developmental StudiesHybridoma Bank (Iowa City, Iowa). The Oct-4 antibody was purchased fromBD Biosciences (San Jose, Calif.). Localization of SSEA-1, SSEA-3,SSEA-4, Oct-4, integrin α6, and integrin β1 was performed using theAlexa Fluor 488-conjugated anti-mouse antibody (Molecular Probes,Eugene, Oreg.), the Alexa Fluor 568-conjugated anti-mouse antibody(Molecular Probes), and the DakoCytomation kit (DakoCytomation,Carpinteria, Calif.).

In Vitro and In Vivo Differentiation

To confirm spontaneous differentiation in vitro, the colony-formingcells were treated with 0.04% (v/v) trypsin-EDTA (Gibco Invitrogen), andthe dissociated cells were subsequently transferred to 100-mm plasticPetri dishes that contained LIF-free DMEM (Gibco Invitrogen) that wassupplemented with 15% (v/v) FBS (HyClone). The cells were grown untilthe EBs formed. The EBs were seeded separately into 96-well cultureplates and cultured for 7 days. The EBs were stained with the followingspecific markers for the three germ layers: nestin (Santa CruzBiotechnology, Santa Cruz, Calif.) and S-100 (Biodesign International,Saco, Me.) for ectodermal cells; muscle actin (Biodesign International)and desmin (Santa Cruz Biotechnology) for mesodermal cells; andα-fetoprotein (Biodesign International) and troma-1 (Hybridoma Bank) forendodermal cells. Antibody localization was performed with theDakoCytomation kit (DakoCytomation).

To confirm in vivo differentiation, 1×10⁷ colony-forming cells retrievedat the 20^(th) subpassage were injected subcutaneously into adultNOD-SCID mice. Teratomas that formed in the subcutaneous region werecollected 8 weeks post-transplantation and fixed with 4% (v/v)paraformaldehyde (Sigma-Aldrich). After embedding in paraffin blocks,the tissues were stained with hematoxylin and eosin for examinationunder a phase-contrast microscope (BX51TF; Olympus, Kogaku, Japan).

Induction of Differentiation into Neuronal Cells

For in vitro differentiation into neuronal lineage cells, thecolony-forming cells were dissociated and plated onto a 0.1%gelatin-coated plastic culture dish at a density of 0.5-1.5×10⁴/cm² inmodified N2B27 medium that consisted of DMEM/F12 (Gibco Invitrogen)supplemented with N2 (Gibco Invitrogen) and B27 (Gibco Invitrogen).Morphological evaluation was conducted throughout the culture period andthe culture medium was changed at intervals of 2 days. Differentiatedcells were maintained by replating into fibronectin-coated tissueculture dishes. Immunohistochemical analysis was conducted subsequently.Differentiated cells were fixed with 4% (v/w) paraformaldehyde(Sigma-Aldrich) for 5 min, incubated in blocking solution (PBSsupplemented with 5% FBS), and the fixed cells were reacted with primaryantibodies directed against nestin (Santa Cruz Biotechnology), β-tubulintype III (Chemicon), O4 (Chemicon), and glial fibrillary acidic protein(GFAP; Chemicon). The antigen-antibody complexes were visualized byreacting with the following fluorescent secondary antibodies: AlexaFluor 488-conjugated anti-goat (Molecular Probes); Alexa Fluor568-conjugated anti-mouse (Molecular Probes); and Alexa Fluor488-conjugated anti-mouse (Molecular Probes). The stained cells wereobserved under a laser scanning confocal microscope with a krypton-argonmixed gas laser excitation at 488 nm or 568 nm and using the fluoresceinfilter (Bio-Rad, Hemel Hempstead, UK).

Primer Design

The Primary3 software (Whitehead Institute/MIT Center for GenomeResearch) was used to design all the specific primers used in theseexperiments. All the PCR primers were designed based on mouse cDNA andgenomic DNA sequences obtained from GenBank. The specificities of thedesigned primers were tested by conducting 40 PCR cycles of 95° C. for30 sec, the annealing temperature (shown in table S6) for 45 sec, and72° C. for 30 sec. The primer sequences are listed in Table 1.

TABLE 1 Oligonucleotide primers and PCR cycling conditions GenBankPrimer sequence Size Genes number Sense (5′ > 3′) Anti-sense (5′ > 3′)(bp) Temp β-actin (RT) X03672 ACCGTGAAAAGATGACCCAG TCTCAGCTGTGGTGGTGAAG254 60 β-actin (R-T) X03672 TACCACAGGCATTGTGATGG TCTTTGATGTCACGCACGATT200 60 Oct-4 (RT, R-T) M34381 GAAGCCCTCCCTACAGCAGA CAGAGCAGTGACGGGAACAG297 60 Nanog (RT, R-T) AY455282 CCCCACAAGCCTTGGAATTACTCAAATCCCAGCAACCACA 255 60 Rex-1 (RT) M28382 ACATCCTAACCCACGCAAAGTGATTTTCTGCCGTATGCAA 294 60 Rex-1 (R-T) M28382 TCCCCGTGTAACATACACCACTTCGTCCCCTTTGTCATGT 247 60 Cripto (RT) M87321 CTTTAAGCAGGGAGGTGGTGTAAAGCCATCTGCCACAATG 195 60 Cripto (R-T) M87321 CGGAGATCTTGGCTGCTAACCTTCGACGGCTCGTAAAAAC 200 60 Dnmt3b (RT) BC105922 AGTCCATCGCTGTGGGAACTGGGCGGGTATAATTCAGCAA 226 60 Dnmt3b (R-T) BC105922 GTCCGGAAAATCACCAAGAACCAGAAGAATGGACGGTTGT 201 60 Tert (RT) AF051911 GGATCCTGGCTACGTTCCTGTGCCTGACCTCCTCTTGTGA 208 60 Tert (R-T) AF051911 GCAGTGGTCCGGAGAGATAGACACTGTGACGCAGGAAGTG 224 60 Lif Rc (RT, R-T) BC031929GCTGAGTGGTAAAGATACCG TTCGTTGGACTCATACAACA 261 60 Stat3 (RT) AY299489TTTGGAATGAAGGGTACATC CAAATGACATGTTGTTCAGC 228 60 Bmp4 (RT) BC013459TGAGAGACCCCAGCCTAAGA AAACTTGCTGGAAAGGCTCA 259 60 Fgf4 (RT) BC104312CAGTCTTCTGGAGCTCTCTC AGGAAGTGGGTTACCTTCAT 282 60 Foxd3 (RT) AF067421CAAGAACAGCCTGGTGAAG GTCCAGGGTCCAGTAGTTG 262 60 Sox2 (RT) AB108673ACGCTCATGAAGAAGGATAA GTAGGACATGCTGTAGGTGG 345 60 CD9 (RT) U60473ATGCTACCACTGTTTCCAAC ACAAGTTAAACTGGCAGCAT 212 60 Gdf3 (RT) BC101963CGAGTTTCAAGACTCTGACC TAGAGGACCTTCTGGAGACA 276 60 Ncam (R-T) Y00051AGATGGTCAGTTGCTGCCAA AGAAGACGGTGTGTCTGCTT 187 60 Nestin (R-T) BC062893TAGAGGTGCAGCAGCTGCAG AGCGATCTGACTCTGTAGAC 170 60 Smooth muscle NM_007392ACTGGGACGACATGGAAAAG CATCTCCAGAGTCCAGCACA 240 60 actin (R-T) Desmin(R-T) NM_010043 TGACAACCTGATAGACGACC TTAAGGAACGCGATCTCCTC 180 60α-fetoprotein (R-T) BC066206 TGCACGAAAATGAGTTTGGGA TTGCAGCCAACACATCGCTA159 60 Troma1 (R-T) D90360 ATCGAGATCACCACCTACCG TCTTCACAACCACAGCCTTG 24160 Zfy1 (gDNA) AC163622 GTTACTCATTTTCAGGTGTTCTGGGGTGTCAGCTGTTATAGGATCAGTGA 572 62 Xist (gDNA) AJ421479.1GAGATACATTTATTTGCTCA GACTTAGTTTGGTTTCTTTA 540 55 RT = ReverseTranscriptase Polymerase Chain Reaction, R-T = Real-Time PolymeraseChain Reaction, gDNA = genomic DNA Polymerase Chain Reaction.

Real-Time PCR Analysis

Total RNA of EBs collected at 7, 14, 21, 28, 35, and 42 days wasextracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, Calif.)according to the manufacturer's instructions. The cDNAs were synthesizedfrom approximately 1 μg of total RNA using the Reverse TranscriptionSystem (Promega, Madison, Wis.). Subsequently, the expression levels ofspecific genes in EBs were quantified by real-time PCR using the DyNAmoHS SYBRGreen qPCR Kit (Finnzymes, Espoo, Finland). PCR amplification wasperformed in a final volume of 25 μl with the ABI PRISM 7700 sequencedetection system (Applied Biosystems, Foster, Calif.) and using thecycling parameters of 2 min at 50° C., 15 min at 95° C., followed by 40cycles of 15 sec at 95° C., 30 sec at 60° C., and 30 sec at 72° C. Thedissociation curve was recorded to check the PCR specificity. The finaloptimized concentration of each primer was 300 nM, and the absence ofinter- and/or intra-molecular duplex formation between primers wasconfirmed in a control real-time PCR reaction that lacked template. ThemRNA level of each gene in each sample was normalized to that ofβ-actin. The relative mRNA level was presented as 2^(−ΔΔCt), whereCt=the threshold cycle for target amplification, ΔCt=Ct_(target gene)(genes specific for the three embryonic germ layercells)−Ct_(internal reference) (β-actin), and ΔΔCt=ΔCt_(sample) (EBscultured for 0, 21, or 35 days)−ΔCt_(calibrator) (EBs cultured for 7days).

Sex Determination by Genomic DNA-PCR Analysis

Total genomic DNA from each established stem cell line was extractedusing the G-spin Genomic DNA Extraction Kit (iNtRON Biotechnology,Seoul, Korea) according to the manufacturer's instruction. The extractedgenomic DNA was subjected to PCR amplification with primers for the Zfy1(Y chromosome-specific) and Xist (X chromosome-specific) genes. The PCRproducts were size-fractionated by 1.2% agarose gel electrophoresis andvisualized by ethidium bromide staining.

Karyotyping and DNA Content Analysis by FACS

For karyotyping, the cells were incubated in medium that contained 0.1μg/ml colcemid (Sigma-Aldrich) for 3 hrs at 37° C. in 5% CO₂ in ahumidified air atmosphere. The treated cells were trypsinized andresuspended for 15 min in 0.075 M KCl (Sigma-Aldrich) at 37° C. Thecells were then placed in a hypotonic solution and subsequently fixed ina 3:1 (v/v) mixture of methanol (Sigma-Aldrich) and acetic acid(Sigma-Aldrich). Chromosomes were spread onto heat-treated slides andstained with Giemsa solution (Gibco Invitrogen). Subsequently, thechromosomes were sorted using the Cytovision (Applied Imaging Co., SantaClara, Calif.).

For FACS analysis to measure DNA content, the harvested cells werewashed in Ca²⁺- and Mg²⁺-free Dulbecco's PBS (DPBS; Gibco Invitrogen)and suspended in 70% (v/v) ethanol (Sigma-Aldrich) for 1 hr at 4° C. Thecells were centrifuged at 390×g for 4 min and resuspended in 0.5 ml ofCa²⁺- and Mg²⁺-free DPBS (Gibco Invitrogen) that contained 0.1 mg/mlribonuclease (Sigma-Aldrich) and 0.1 mg/ml propidium iodide(Sigma-Aldrich). After 30 min at room temperature in the dark, the cellsuspension was analyzed by a Becton Dickinson FACS-Vantage SE (BectonDickinson, San Jose, Calif.) equipped with a two water-cooled laser. Thedata were analyzed using the CELL Quest™ ver. 3.3 software (BectonDickinson).

Telomerase Activity Assay

Telomerase activity was determined using the TRAP_(EZE) TelomeraseDetection Kit (Chemicon) according to manufacturer's instructions. Thesix established lines were analyzed at the 20^(th) subpassage and PCRamplification was conducted for 27 cycles. The PCR product was separatedby electrophoresis in non-denaturing polyacrylamide gels.

Chimera Formation

To confirm pluripotency on a larger scale, 10-15 colony-forming cellsmaintained for different numbers of subpassages (7-13 or 32-39) wereaggregated with 8-cell embryos and, 20 h after aggregation, theblastocysts derived from the aggregated embryos were transferred to theuteri of 2.5-dpc pseudo-pregnant ICR females (S. A. Wood, N. D. Allen,J. Rossant, A. Auerbach, A. Nagy, Nature 365, 87 (1993)).

DNA Microsatellite Analysis

DNA microsatellite analysis was performed with genomic DNA samples fromthe E14 ES cells, B6D2F1 MEFs, B6CBAF1 tail, newly established ESC-likecells, and teratomas induced from established ESC-like cells. Threespecific mouse microsatellite primers (D03Mit200, D11Mit4, andD15Mit159) were used (http://www.cidr.jhmi.edu/mouse/mmset.html). Thegenomic DNA from each sample was amplified by PCR for the threemicrosatellite loci. Forward primers were synthesized with a fluorescenttag (FAM, TET, or HEX) at the 5′-end, and fluorescent PCR amplificationwas performed with the PC808 program TEMP control system (ASTEC). ThePCR products were subsequently analyzed in the ABI Prism 310 DNAautomated sequencer (Applied Biosystems). Digital images were obtainedusing the Genescan Data Collection ver. 2.5 software (AppliedBiosystems). Each fluorescent peak was quantified for base-pair size,peak height, and peak area.

Immunostaining of ESC-Like Cells, MEFs, and Mitotically Inactivated MEFs

The colony-forming ES cells and MEF with or without mitomycin treatmentwere fixed in 4% (v/v) formaldehyde (Sigma-Aldrich) at room temperaturefor 10 min. The fixed ESC-like cells were exposed to antibodies directedagainst Vasa (Abcam, Cambridge, UK), Fragilis (Abcam), and AMH (Abcam).The localization of Vasa, Fragilis, and AMH was detected using the AlexaFluor 488-conjugated anti-rabbit antibody (Molecular Probes) and theDakoCytomation kit (DakoCytomation). Furthermore, the fixed MEFs andmitotically inactivated MEFs were exposed to antibodies directed againstOct-4 (BD Biosciences), Nanog (Abcam), Vasa (Abcam), Fragilis (Abcam),CD44 (Chemicon), CD45 (Chemicon) and AMH (Abcam). Antibody localizationwas performed with the DakoCytomation kit (DakoCytomation).

Immunofluorescence of Ovarian Tissue-Dissociated Cells and ESC-LikeCells

The dissociated ovarian cells were fixated in 70% ethanol for 1 hr at 4°C. The fixed cells were centrifuged at 390×g for 4 min and washed twicewith 0.5 ml of Ca²⁺- and Mg²⁺-free DPBS (Gibco Invitrogen) thatcontained 2% (v/v) FBS (HyClone). The fixed ovarian tissue-dissociatedcells were reacted for 1 hr at 4° C. with primary antibodies directedagainst Oct-4 (BD Biosciences), Nanog (Abcam), Vasa (Abcam), CD44(Chemicon), and AMH (Abcam). Moreover, the ESC-like cells dissociated by1 mM EDTA (BIONEER, Seoul, Korea) were reacted for 1 hr at roomtemperature with primary antibodies to PE-conjugated Sca-1 (BDBiosciences), FITC-conjugated CD44 (BD Biosciences), biotin-conjugatedCD34 (BD Biosciences) and biotin-conjugated CD45 (BD Biosciences). Sca-1and CD44 were specific markers for MSCs, while CD34 and CD45 were usedfor epithelial stem cell-specific and hematopoietic stem cell-specificmarkers, respectively. After washing twice, the antigen-antibodycomplexes were visualized with the following fluorescent secondaryantibodies: Alexa Fluor 488-conjugated anti-mouse (Molecular Probes),Alexa Fluor 488-conjugated anti-rabbit (Molecular Probes), Alexa Fluor568-conjugated anti-mouse (Molecular Probes), Alexa Fluor 568-conjugatedanti-rat (Molecular Probes), and Streptavidin-phycoerythrin (SAv-PE, BDBiosciences). The stained ovarian tissue-dissociated cells were observedunder a laser scanning confocal microscope (Bio-Rad), and the stainedESC-like cells were analyzed by flow cytometry (FACSCALIBUR, BectonDickinson).

Analysis of Imprinted Genes

Bisulfite genomic sequencing of the differentially methylated region 2(DMR2) of Igf2 was carried out as described previously (S. Sato, T.Yoshimizu, E. Sato, Y. Matsui, Mol. Reprod. Dev. 65, 41 (2003)). PCRamplification of each DMR2 region of the bisulfite-treated genomic DNAswas carried out using specific primers: Forward (1088-1107 bp)5′-GTTGGGGATATGTGATATTTA-3′, Reverse (1307-1330 bp)5′-AAACCATAACCTTTAACCTCTCTA-3′. The nucleotide positions of primersreferred to Igf2 DMR2 sequence (GenBank accession no. AY849922). Fourregions (P1, P2, P3 and P4) in DMR2 are the position to analyzemethylation. The cytosines of the CpGs are located at the followingpositions: 1227, 1229, 1234 and 1240 (GenBank accession no. AY849922).

Deposit of Somatic Cell-Derived ESC-Like Cell

Of the fibroblast-derived ESC-like cells showing all of the ES cellcharacteristics described above, one cell was named “tScB6CD-SNU-1” anddeposited on May 16, 2006 in the International Depository Authority, theKorean Cell Line Research Foundation and was given accession No.KCLRF-BP-00135.

Results

We have continuously sought alternative techniques. During preparationof mouse embryonic fibroblasts (MEFs), we occasionally foundcolony-like, homogenous cell mass. Whereas, we detected embryonic stemcell (ESC)-specific Oct-4 and Nanog expression in medullar tissue nearthe blood vessels and the theca cell region of the ovarian follicles(FIG. 1). We further knew that cells dissociated from the ovariescontain ESC-, mesenchymal stem cell (MSC)-, germ cell- and/or follicularcell-specific marker-positive cells. First, we cultured the B6D2F1embryonic fibroblasts alone or cultured dissociated ovarian cells ofless than 40 μm in diameter (B6CBAF1) without fibroblasts in Dulbecco'sminimal essential medium (DMEM) that contained 5,000 U/ml leukemiainhibitory factor (LIF). However, neither of these cultures yieldedcolonies (Table 2).

TABLE 2 Culturing of the mouse embryonic fibroblasts (MEF) used forprimary culture with ovarian cells (B6D2F1; C57BL/6 × DBA2) orsubpassage of colony-forming cells (ICR strain), and culturing ofovarian tissue cells with or without MEF feeder cells in medium ^(a)supplemented with leukemia inhibitory factor Strain of MEF With(+)/without (−) Colony Trials Culture of feeder mitomycin treatmentformation 1 MEF only ICR + No 2 MEF only ICR + No 3 MEF only ICR + No 4MEF only ICR + No 5 MEF only ICR + No 6 MEF only ICR + No 1 MEF only ICR− No 2 MEF only ICR − No 3 MEF only ICR − No 4 MEF only ICR − No 5 MEFonly ICR − No 6 MEF only ICR − No 1 MEF only B6D2F1 + No 2 MEF onlyB6D2F1 + No 3 MEF only B6D2F1 + No 4 MEF only B6D2F1 + No 5 MEF onlyB6D2F1 + No 6 MEF only B6D2F1 + No 1 MEF only B6D2F1 − No 2 MEF onlyB6D2F1 − No 3 MEF only B6D2F1 − No 4 MEF only B6D2F1 − No 5 MEF onlyB6D2F1 − No 6 MEF only B6D2F1 − No 1 Ovarian cell N/A N/A No only 2Ovarian cell N/A N/A No only 3 Ovarian cell N/A N/A No only 4 Ovariancell N/A N/A No only 5 Ovarian cell N/A N/A No only 6 Ovarian cell N/AN/A No only N/A = Not Analysis ^(a)DMEM supplemented withβ-mercaptoethanol, non-essential amino acids, L-glutamine, lyophilizedmixture of penicillin and streptomycin and fetal bovine serum wasemployed as a based medium.

We then cocultured the B6D2F1 fibroblasts with ovarian cells in DMEM, towhich 1,000, 2,000 or 5,000 U/ml LIF was added. Colony-like cellaggregations were observed (Table 3), and, in total, 6/24 trials (25%)yielded colonies on day 7 of primary culture in 5,000 U/mlLIF-containing medium (FIG. 2). These colonies have been maintained formore than 6 months with 65 passages and have been stored in liquidnitrogen at −196° C.

TABLE 3 Outcome of culturing mouse embryonic fibroblasts^(a,b) withovarian tissue cells in media supplemented with different concentrationsof leukemia inhibitory factor (LIF) LIF conc., in medium^(c) ColonyTrials (units/ml) formation^(d) Established cell line^(d) 1 1,000 No — 22,000 No — 2,000 No — 3 5,000 Yes Yes (tScB6CD-SNU-1) 4 5,000 No — 55,000 No — 5,000 No — 6 5,000 No — 5,000 No — 5,000 No — 7 5,000 No —5,000 No — 5,000 No — 5,000 No — 5,000 No — 8 5,000 No — 5,000 Yes Yes(tScB6CD-SNU-2) 9 5,000 Yes Yes (tScB6CD-SNU-3) 5,000 Yes Yes(tScB6CD-SNU-4) 5,000 No — 5,000 Yes Yes (tScB6CD-SNU-5) 5,000 No —5,000 Yes Yes (tScB6CD-SNU-6) 5,000 No — 10 5,000 No — 5,000 No — 5,000No — ^(a)The embryonic fibroblast monolayer treated with mitomycin C wasprovided for cell culture. ^(b)Retrieved from the 13.5-day-old fetusesin mated C57BL/6 females with DBA2 male ^(c)Collected from 8-week-old,adult female F1 mice (B6CBAF1; C57BL/6 × CBA/Ca) ^(d)DMEM supplementedwith β-mercaptoethanol, non-essential amino acids, L-glutamine,lyophilized mixture of penicillin and streptomycin and fetal bovineserum was employed as a based medium.

Consequently, we attempted to clarify the progenitor cells of theestablished colony-forming cells. Short tandem repeat (STR)microsatellite analysis using three markers was undertaken todistinguish the genotypes of the ovarian cell donor, the fibroblasts andthe control ESCs (129/Ola strain). The microsatellite loci of theestablished cells matched perfectly with those of the B6D2F1 fibroblastmonolayer, and were completely different from those of the ovary donorsand E14 ES cells (Tables 4a and 4b).

TABLE 4a Short-tandom repeat microsatellite analysis of establishedembryonic stem cell-like cells, teratomas, and fibroblast donor stainsD3Mit200^(a) D11Mit4^(a) Sample Size 1 Size 2 Sample Size 1 Size 2 Size3 Ovary 124.36 — Ovary donor 248.72 295.13 — donor Feeder 101.09 124.29Feeder cell^(b) 248.75 285.14 — cell^(b) E14 124.36 — E14 248.92 293.22295.33 tScB6CD- 101.16 124.31 tScB6CD- 248.98 285.09 — SNU-1 SNU-1tScB6CD- 101.09 124.31 tScB6CD- 248.93 285.14 — SNU-2 SNU-2 tScB6CD-101.09 124.34 tScB6CD- 248.88 285.30 — SNU-3 SNU-3 tScB6CD- 101.03124.20 tScB6CD- 248.66 285.08 — SNU-4 SNU-4 tScB6CD- 101.24 124.41tScB6CD- 248.87 285.08 — SNU-5 SNU-5 tScB6CD- 101.17 124.31 tScB6CD-248.71 285.10 — SNU-6 SNU-6 Teratoma 101.04 124.17 Teratoma 248.91285.34 —

TABLE 4b Short-tandom repeat microsatellite analysis of establishedembryonic stem cell-like cells, teratomas, and fibroblast donor stainsD15Mit159^(a) Sample Size 1 Size 2 Ovary donor 137.39 139.43 Feedercell^(b) 111.76 137.41 EH 139.45 141.62 tScB6CD-SNU-1 111.97 137.54tScB6CD-SNU-2 110.79 137.41 tScB6CD-SNU-3 110.76 139.45 tScB6CD-SNU-4111.85 137.45 tScB6CD-SNU-5 111.76 137.42 tScB6CD-SNU-6 111.79 137.41Teratoma 110.83 138.50

None of the cells in the fibroblast monolayer were not positive forspecific markers of ESCs, germ cells, follicular cells, MSCs, andhematopoietic stem cells (FIG. 3). From these results, we conclude thatMEFs are the origin of the established cells. The ovarian niche, whichincludes ovarian tissue-specific stem cells and germ cells, reprogramsterminally-differentiated fibroblasts to acquire sternness. Wecontinuously attempt to further establish the colony-forming cells fromthe fibroblasts of different strains and two more lines were derivedfrom ICR mice.

Next, we characterized the established colony-forming cells. All sixlines had the XX sex chromosomes (FIG. 4). The karyotype of thecolony-forming cells collected from early (6-7) or late (30) passageshad the tetraploid karyotype, while no tetraploid cells were found inovarian cells and MEFs. The colony-forming cells retrieved at the20^(th) subpassage were positive for ESC-specific markers and expressedESC-specific genes (FIGS. 5 a and 5 b). They had telomerase activity(FIG. 6) and 26-47% of the imprinted gene Igf2 in the established lineswas methylated (Table 5).

TABLE 5 Bisulfite genomic sequencing analysis of the DMR2 of Igf2 inembryonic stem cell (ESC)-like cells of 6 lines and E14 cells of thecontrol Mean % (±SD) of methylation Cell lines P1 P2 P3 P4 E14 52.19 ±2.81 39.15 ± 2.71 39.04 ± 6.91 48.91 ± 4.79 tScB6CD-SNU-1 48.64 ± 1.6231.45 ± 1.57 34.24 ± 2.28 46.51 ± 2.36 tScB6CD-SNU-2 33.25 ± 1.38 24.74± 0.81 21.98 ± 1.28 33.43 ± 2.72 tScB6CD-SNU-3 39.08 ± 4.27 23.22 ± 1.8324.94 ± 2.94 41.09 ± 2.77 tScB6CD-SNU-4 47.51 ± 4.68 27.82 ± 1.57 27.87± 3.36 46.94 ± 2.87 tScB6CD-SNU-5 36.94 ± 4.82  30.9 ± 3.72 31.25 ± 4.7940.79 ± 4.79 tScB6CD-SNU-6 40.36 ± 0.73 26.82 ± 2.69 28.57 ± 2.43 43.82± 3.33

Neither the established cells nor the E14 ES cells were positive fortissue-specific stem cell markers (Sca-1, CD44, AMH, Vasa and Fragilis;FIGS. 7-8). When the colony-forming cells were cultured in LIF-freemedium, the cells formed embryoid bodies (EBs; FIG. 9) and subcutaneoustransplantation of the colony-forming cells into NOD-SCID mice formedteratomas (FIG. 10). The colony-forming cells differentiated intoneuronal cells in a spontaneous manner after being treated with N2B27solution. To further verify pluripotency, the colony-forming cellscollected at early (7-13 passages) and late (32-39 passages) subpassageswere aggregated with 8-cell embryos, and the blastocysts derived fromthe aggregated embryos were transferred into the uteri of surrogatemothers. In all, 74 offspring were delivered, nine (12.2%) of which weresomatic chimeras (Table 6).

TABLE 6 Production of somatic chimeras developed from transplantedblastocysts generated from embryos aggregated with established embryonicstem cell (ESC)-like cells Aggregated No. of No. (%)^(b) of somaticchimeras stem cells Embryos Offsprings Live Live subcultured transferredRecipient delivered^(a) Total males females Dead  7 to 13 times 573 4648  9 (19) 1 3 5 32 to 39 times 387 27 26 0 (0) 0 0 0 The establishedcell line of ScB6CD-SNU-1 was used for aggregation with 8-cell embryosof ICR strain. ^(a)In some cases, fetuses were delivered by cesareansection at 19.5 dpc, and transferred to nursing female mice.^(b)Percentage of the number of offsprings delivered.

Four of the chimeras lived (3 females and 1 male; FIG. 11). Theseresults show that the established tetraploid cells are pluripotent stemcells, with almost similar properties as diploid ES cells (Tables 7a and7b).

TABLE 7a Summary of the characterization of the embryonic stem cell(ESC)-like cell lines derived from coculturing of adult ovarian cellsand embryonic fibroblasts in mice Stem cell specific markers EstablishedIntegrin Integrin ES cell line Karyotype AP SSEA-1 SSEA-3 SSEA-4 Oct-4α6 β1 tScB6CD- Tetraploid, + + − − + + + SNU-1 XX tScB6CD-Tetraploid, + + − − + + + SNU-2 XX tScB6CD- Tetraploid, + + − − + + +SNU-3 XX tScB6CD- Tetraploid, + + − − + + + SNU-4 XX tScB6CD-Tetraploid, + + − − + + + SNU-5 XX tScB6CD- Tetraploid, + + − − + + +SNU-6 XX

TABLE 7b Summary of the characterization of the embryonic stem cell(ESC)-like cell lines derived from coculturing of adult ovarian cellsand embryonic fibroblasts in mice Methylation Formation of Established %of IgF2 Telomerase Neuronal cell Embryoid ES cell line Karyotype (Mean ±SD) activity differentiation Teratoma body tScB6CD-SNU-1 Tetraploid,40.2 ± 1.5 + Yes Yes Yes XX tScB6CD-SNU-2 Tetraploid, 28.4 ± 1.3 + YesYes Yes XX tScB6CD-SNU-3 Tetraploid, 32.1 ± 1.8 + Yes Burging Yes XXtScB6CD-SNU-4 Tetraploid, 37.5 ± 1.8 + Yes Burging Yes XX tScB6CD-SNU-5Tetraploid, 35.0 ± 1.7 + Yes Burging Yes XX tScB6CD-SNU-6 Tetraploid,34.9 ± 1.1 + Yes Burging Yes XXWe evaluated whether the tetraploid ESC-like cells can restore thenormal diploid karyotype after differentiation. No diploid cells werepresent among the established stem cells, while there were no tetraploidcells in the teratomas (FIG. 12). The population of diploid cellsgradually increased as the EB was formed and the diploid cells finallypredominated in the EBs collected from 21 days post-treatment. Theresults of the STR microsatellite analysis show that the genotype of thedifferentiated cells in the teratomas matched that of the feeder celldonor (Table 4). From these results, diploid-to-tetraploid andtetraploid-to-diploid shifts occur during the acquisition of sternnessby reprogramming and dedifferentiation and during differentiation intosomatic cells, respectively, which do not affect the genotype of theestablished cells.

Examples For ESC-Like Cells Derived from Ovarian Cells Materials andMethods Animals

Animals provided for this study were bred at the Laboratory of Gameteand Stem Cell Biotechnology, Seoul National University, Korea. They weremaintained under controlled conditions of lighting (14 L:10 D),temperature (20-22° C.), and humidity (40-60%). Female F1 hybrid(B6D2F1) mice were produced by mating female C57BL/6 mice with male DBA2mice and 8-week-old female mice were used as the donors of the ovaries.All of the procedures for animal management, breeding, and surgeryfollowed the standard operation protocols of Seoul National University,and the review board of Experimental Animal Resources, Seoul NationalUniversity, approved the usage of animals and the relevant experimentalprocedures (approval no. SNU-050331-2). Experimental samples wereproperly managed, and quality control of the laboratory facility andequipment was conducted.

In Situ Hybridization

An in situ hybridization detection system kit (DF132-60K; BioGenex, SanRamon, Calif.) and probe (Biognostik, Gottingen, Germany) were usedaccording to the supplied protocol, which was optimized to minimizebackground signals. The collected ovarian tissues of C57BL/6 or B6D2F1(C57BL/6×DBA2) were frozen in optimal cutting temperature compound(Tissue-Tek OCT compound; Sakura, Torrance, Calif.) at −70° C. Thecryo-samples were cut at 10-μm thickness and fixed at room temperaturefor 5 min in PBS that contained 4% (v/v) formaldehyde (Sigma-Aldrich).The slides were dehydrated in an ethanol series (70%, 80%, 90%, 95%, and100%). The air-dried samples were prehybridized in HybriBuffer-ISH at30° C. for 3 h. Hybridization was conducted in HybriBuffer-ISH thatcontained the Oct-4 or Nanog HybriProbe at 40° C. overnight. The slideswere washed in 1× sodium chloride/sodium citrate solution (SSC) at roomtemperature for 5 min, in 0.1×SSC at 45° C. for 15 min, and in 1×PBSsupplemented with 0.1% (v/v) Tween-20 (USB, Cleveland, Ohio) at roomtemperature for 3 min. The slides were incubated in Power Block Reagentat room temperature for 10 min and with the biotinylatedanti-fluorescein antibody at room temperature for 40 min. After washingwith 1×PBS supplemented with 0.1% (v/v) Tween-20 (USB), the slides wereincubated in streptavidin-alkaline phosphatase conjugate at roomtemperature for 20 min, and washed with 1×PBS supplemented with 0.1%(v/v) Tween-20 (USB). Alkaline phosphatase was activated by incubatingwith Activation Buffer at room temperature for 1 min. The slides weredeveloped with NBT/BCIP at room temperature for 15 min, mounted withSupermount, and observed under a phase-contrast microscope (BX51TF;Olympus).

Preparation of MEFs

Outbred (ICR) 13.5-day-old fetuses were euthanized for the derivation ofMEFs for primary culture and subculture. Embryonic fibroblasts werecollected from the fetuses, and the visceral organs, head, andextremities of the fetuses were removed. The remaining tissue was cutinto small pieces and was subsequently incubated in 0.04% (v/v)trypsin-EDTA (Gibco Invitrogen, Grand Island, N.Y.) for 6 min withagitation at 37° C. After being centrifuged at 110×g for 2 min, thesupernatants were diluted in 10% (v/v) fetal bovine serum (FBS; HyCloneLaboratories, Logan, Utah)-containing DMEM medium (Gibco Invitrogen) andcentrifuged at 390×g for 4 min. The pellets of the collected fibroblastswere suspended and replaced in DMEM medium for monolayer formation. Whenthe fibroblasts formed a confluent monolayer, they were frozen in 10%dimethylsulfoxide (Gibco Invitrogen).

Ovarian Cell Preparation

The ovaries were collected and, after the removal of adherent tissue,the retrieved tissue was chopped using a surgical blade. The specimenswere incubated initially for 30 min in a dissociation medium thatconsisted of a 50:50 (v:v) mixture of 0.25% (v/w) trypsin-EDTA (GibcoInvitrogen) and DMEM (Gibco Invitrogen) that was supplemented with 750units/ml collagenase type I (Sigma-Aldrich, St. Louis, Mo.) and 0.03%(v/v) fetal bovine serum (FBS; HyClone), at 37° C. The dissociated cellswere filtered through a 40-μm cell strainer (BD Falcon, Franklin Lakes,N.J.) and centrifuged at 390×g for 4 min. They were initially seededinto 60 mm×10 mm culture dishes. The stromal cells mixed in with thedissociated cells were removed 30 min after initial seeding and thebuoyant cells present above the bottomed stromal cells were re-seededonto MEF monolayers in the dishes. In some replications, the filteredcells were directly seeded onto MEF monolayer without stromal cellremoval. The culture medium was DMEM medium that was supplemented with0.1 mM β-mercaptoethanol (Gibco Invitrogen), 1% (v/v) nonessential aminoacids (Gibco Invitrogen), 2 mM L-glutamine (Sigma-Aldrich), 1% (v/v)lyophilized mixture of penicillin and streptomycin (Gibco Invitrogen),5,000 units/ml mouse LIF (Chemicon, Temecula, Calif.), and 15% (v/v) FBS(HyClone).

Coculture of MEFs and Ovarian Cells for Establishing Colony-FormingCells

MEF monolayers were treated with 10 μg/ml mitomycin C (Sigma-AldrichCorp.) for 3 h in gelatin-coated 60-mm tissue culture dishes andsubsequently used for establishing colony-forming cells. The ovariancells prepared were additionally seeded into the dishes that containedMEF monolayers and cultured at 37° C. under 5% CO₂ in a humidified airatmosphere. On day 4 of culture, the ovarian cells reached 60-70% ofconfluency were subsequently replated on a new monolayer in the samesize culture dishes containing culture medium. The LIF concentrationadded to the culture medium ranged from 1,000 to 5,000 units/ml inprimary culture and was fixed at 1,000 units/ml for the subcultures. Atthe end of the primary culture (on day 7 of culture), colony-formingcells were mechanically removed with a capillary pipette and subculturedwith the MEF monolayer at intervals of 3 days, whereas the medium waschanged daily.

Marker Staining of Colony-Forming Cells

For characterization using stem cell-specific markers, colony-formingcells collected at the 20^(th) subpassage were fixed in 4% (v/v)formaldehyde (Sigma-Aldrich) at room temperature for 10 min. Thereactivity of the colony-forming cells to alkaline phosphatase wasassessed with Fast Red TR/naphthol AS-MX phosphate (Sigma-Aldrich).Antibodies against Oct-4 (BD Biosciences, San Jose, Calif.),stage-specific embryonic antigens (SSEA)-1 (Developmental StudiesHybridoma Bank, Iowa City, Iowa), SSEA-3 (Developmental StudiesHybridoma Bank), SSEA-4 (Developmental Studies Hybridoma Bank), integrinα6 (Santa Cruz Biotechnolgy, Santa Cruz, Calif.), integrin β1 (SantaCruz Biotechnolgy), Vasa (Abcam, Cambridge, UK), Fragilis (Abcam) andAMH (Abcam) were provided for the marker staining. Localization ofSSEA-1, SSEA-3, SSEA-4, Oct-4, integrin α6, integrin β1, Vasa, Fragilisand AMH was performed using the Alexa Fluor 488-conjugated anti-mouseantibody (Molecular Probes, Eugene, Oreg.), the Alexa Fluor568-conjugated anti-mouse antibody (Molecular Probes), and theDakoCytomation kit (DakoCytomation, Carpinteria, Calif.).

In Vitro and In Vivo Differentiation

To confirm spontaneous differentiation in vitro, the colony-formingcells were treated with 0.04% (v/v) trypsin-EDTA (Gibco Invitrogen), andthe dissociated cells were subsequently transferred to 100-mm plasticpetri dishes that contained LIF-free DMEM (Gibco Invitrogen) that wassupplemented with 10% (v/v) FBS (HyClone). The cells were grown untilthe embryoid bodies formed. The embryoid bodies were seeded separatelyinto 4-well culture plates and cultured for 10 to 14 days. The embryoidbodies were stained with the following specific markers for the threegerm layers: nestin (Santa Cruz Biotechnology) and S-100 (Abcam) forectodermal cells; α-smooth muscle actin (Abcam) and desmin (Santa CruzBiotechnology) for mesodermal cells; and α-fetoprotein (BiodesignInternational) and troma-1 (Hybridoma Bank) for endodermal cells.Antibody localization was performed with the DakoCytomation kit(DakoCytomation).

To confirm in vivo differentiation, 1×10⁷ colony-forming cells retrievedat the 20^(th) subpassage were injected subcutaneously into adultNOD-SCID mice. Teratomas that formed in the subcutaneous region werecollected 6 weeks post-transplantation and fixed with 4% (v/v)paraformaldehyde (Sigma-Aldrich). After embedding in paraffin blocks,the tissues were stained with hematoxylin and eosin for examinationunder a phase-contrast microscope (BX51TF; Olympus, Kogaku, Japan).

Induction of Differentiation into Neuronal Cells

For in vitro differentiation into neuronal lineage cells, thecolony-forming cells were dissociated and plated onto a 0.1%gelatin-coated plastic culture dish at a density of 0.5-1.5×10⁴/cm² inmodified N2B27 medium that consisted of DMEM/F12 (Gibco Invitrogen)supplemented with N2 (Gibco Invitrogen) and B27 (Gibco Invitrogen).Morphological evaluation was conducted throughout the culture period andthe culture medium was changed at intervals of 2 days. Differentiatedcells were maintained by replating into fibronectin-coated tissueculture dishes. Immunocytochemical analysis was conducted subsequently.Differentiated cells were fixed with 4% (v/w) paraformaldehyde(Sigma-Aldrich) for 5 min, incubated in blocking solution (PBSsupplemented with 5% FBS), and the fixed cells were reacted with primaryantibodies directed against nestin (Santa Cruz Biotechnology), β-tubulintype III (Chemicon), O4 (Chemicon), and glial fibrillary acidic protein(GFAP; Chemicon). The antigen-antibody complexes were visualized byreacting with the following fluorescent secondary antibodies: AlexaFluor 488-conjugated anti-goat (Molecular Probes); Alexa Fluor568-conjugated anti-mouse (Molecular Probes); and Alexa Fluor488-conjugated anti-mouse (Molecular Probes). The stained cells wereobserved under a laser scanning confocal microscope with a krypton-argonmixed gas laser excitation at 488 nm or 568 nm and using the fluoresceinfilter (Bio-Rad, Hemel Hempstead, UK).

Primer Design

The Primary3 software (Whitehead Institute/MIT Center for GenomeResearch) was used to design all the specific primers used in theseexperiments. All the PCR primers were designed based on mouse cDNA andgenomic DNA sequences obtained from GenBank. The specificities of thedesigned primers were tested by conducting 40 PCR cycles of 95° C. for30 sec, the annealing temperature (shown in table S6) for 45 sec, and72° C. for 30 sec. The primer sequences are listed in SI Table 1.

Reverse Transcriptase-PCR Analysis

Total RNA of the ovaries, brain, heart, lung, liver, stomach, spleen,small intestine, bladder, kidney and skin of C57BL/6 or B6D2F1, andcolony-forming cells was extracted using the RNeasy Plus Mini Kit(Qiagen, Valencia, Calif.) according to the manufacturer's instructions.The cDNAs were synthesized from approximately 1 μg of total RNA usingthe Reverse Transcription System (Promega, Madison, Wis.) and subjectedto PCR amplification with the specific primers. The PCR products weresize-fractionated by 1.2% agarose gel electrophoresis and werevisualized by ethidium bromide staining.

Real-Time PCR Analysis

Total RNA was extracted from the ovaries using the RNeasy Plus Mini Kit(Qiagen, Valencia, Calif.) according to the manufacturer's instructions.The cDNAs were synthesized by the same method used for RT-PCR.Subsequently, the expression levels of specific genes in the ovarieswere quantified using the DyNAmo HS SYBRGreen qPCR Kit (Finnzymes,Espoo, Finland). PCR amplification was performed in a final volume of 25μl with the ABI PRISM 7700 sequence detection system (AppliedBiosystems, Foster, Calif.) and using the cycling parameters of 2 min at50° C., 15 min at 95° C., followed by 40 cycles of 15 sec at 95° C., 30sec at 60° C., and 30 sec at 72° C. The dissociation curve was recordedto check the PCR specificity. The final optimized concentration of eachprimer was 300 nM, and the absence of inter- and/or intra-molecularduplex formation between primers was confirmed in a control real-timePCR reaction that lacked template. The mRNA level of each gene in eachsample was normalized to that of β-actin. The relative mRNA level waspresented as 2^(−ΔΔCt), where Ct=the threshold cycle for targetamplification, ΔCt=Ct_(target gene)−Ct_(internal reference), andΔΔCt=ΔCt_(sample)−ΔCt_(calibrator).

Karyotyping and DNA Content Analysis by FACS

Karyotype of the cell lines was analyzed after they were maintained inculture for 7-8 weeks, which correspond to 16-18 passages. The cellswere treated in culture medium supplemented with 0.05 μg/ml colcemid(Wako) for 1-2 h and were subsequently harvested for trypsinization. Thecells were treated with 0.56% KCl solution for 15 min and fixed in acold methano-acetic acid (3:1) mixture for 30 min on ice. The fixativesolution was changed twice by centrifuging the cells at a 30 mininterval. Chromosomes were spread onto heat-treated slides. A modifiedmethod of Seabright¹⁴ was used for G-banding of air-dried chromosomes.The chromosome spread on glass slides was aged for about a week at roomtemperature, dipped in a 0.025% trypsin solution for 10 sec, rinsed indistilled water and stained with Giemsa's solution in phosphate buffer(pH 6.8) for 10 min. After being washed in distilled water andair-dried, at least 50 spreads were counted for chromosome number and 10banding patterns were analyzed at 300-500 bands resolution.

For FACS analysis to measure DNA content, the harvested cells werewashed in Ca²⁺- and Mg²⁺-free Dulbecco's PBS (DPBS; Gibco Invitrogen)and suspended in 70% (v/v) ethanol (Sigma-Aldrich) for 1 h at 4° C. Thecells were centrifuged at 390×g for 4 min and resuspended in 0.5 ml ofCa²⁺- and Mg²⁺-free DPBS (Gibco Invitrogen) that contained 0.1 mg/mlribonuclease (Sigma-Aldrich) and 0.1 mg/ml propidium iodide(Sigma-Aldrich). After 30 min at room temperature in the dark, the cellsuspension was analyzed by a Becton Dickinson FACS-Vantage SE (BectonDickinson, San Jose, Calif.) equipped with a two water-cooled laser. Thedata were analyzed using the CELL Quest™ ver. 3.3 software (BectonDickinson).

Sex Determination by Genomic DNA-PCR Analysis

Total genomic DNA from each established stem cell line was extractedusing the G-spin Genomic DNA Extraction Kit (iNtRON Biotechnology,Seoul, Korea) according to the manufacturer's instruction. The extractedgenomic DNA was subjected to PCR amplification with primers for the Zfy1(Y chromosome-specific) and Xist (X chromosome-specific) genes. The PCRproducts were size-fractionated by 1.2% agarose gel electrophoresis andvisualized by ethidium bromide staining.

Telomerase Activity Assay

Telomerase activity was determined using the TRAP_(EZE) TelomeraseDetection Kit (Chemicon) according to manufacturer's instructions. Thetwo established lines were analyzed at the 20^(th) subpassage and PCRamplification was conducted for 27 cycles. The PCR product was separatedby electrophoresis in non-denaturing polyacrylamide gels.

DNA Microsatellite Analysis

DNA microsatellite analysis was performed with genomic DNA samples fromB6D2F1 tail, ICR MEFs and two lines of newly established colony-formingcells. Two specific mouse microsatellite primers (D03Mit200 and D11Mit4)that were collected from public database(http://www.cidr.jhmi.edu/mouse/mmset.html) were used. The genomic DNAfrom each sample was amplified by PCR for the two microsatellite loci.Forward primers were synthesized with a fluorescent tag (TET or HEX) atthe 5′-end, and fluorescent PCR amplification was performed with thePC808 program TEMP control system (ASTEC). The PCR products weresubsequently analyzed in the ABI Prism 310 DNA automated sequencer(Applied Biosystems). Digital images were obtained using the GenescanData Collection ver. 2.5 software (Applied Biosystems). Each fluorescentpeak was quantified for base-pair size, peak height, and peak area.

Immunostaining of Ovarian Tissue-Dissociated Cells and Colony-FormingCells

The dissociated ovarian, splenal or small intestinal cells were fixatedin 70% ethanol for 1 h at 4° C. The fixed cells were centrifuged at390×g for 4 min and washed twice with 0.5 ml of Ca²⁺- and Mg²⁺-free DPBS(Gibco Invitrogen) that contained 2% (v/v) FBS (HyClone). The fixedovarian tissue-dissociated cells were reacted for 1 h at 4° C. withprimary antibodies directed against Nanog (Abcam), Vasa, CD44 (Chemicon)or AMH and secondary reacted with Oct-4. Moreover, the colony-formingcells dissociated by 1 mM EDTA (BIONEER, Seoul, Korea) were reacted for1 h at room temperature with primary antibodies to PE-conjugated Sca-1(BD Biosciences), FITC-conjugated CD44 (BD Biosciences),biotin-conjugated CD34 (BD Biosciences) and biotin-conjugated CD45 (BDBiosciences). Sca-1 and CD44 were specific markers for mesenchymal stemcells, while CD34 and CD45 were used for epithelial stem cell-specificand hematopoietic stem cell-specific markers, respectively. Afterwashing twice, the antigen-antibody complexes were visualized with thefollowing fluorescent secondary antibodies: Alexa Fluor 488-conjugatedanti-mouse (Molecular Probes), Alexa Fluor 488-conjugated anti-rabbit(Molecular Probes), Alexa Fluor 568-conjugated anti-mouse (MolecularProbes), Alexa Fluor 568-conjugated anti-rat (Molecular Probes), andStreptavidin-phycoerythrin (SAv-PE, BD Biosciences). The stained ovariantissue-dissociated cells were observed under a laser scanning confocalmicroscope (Bio-Rad), and the stained colony-forming cells were analyzedby flow cytometry (FACSCALIBUR, Becton Dickinson).

Deposit of ESC-Like Cell

Of the ovarian cell-derived ESC-like cells showing all of the ES cellcharacteristics described above, one cell was named “OSC-B6D2-SNU-1” anddeposited on Nov. 17, 2006 in the International Depository Authority,the Korean Cell Line Research Foundation and was given accession No.KCLRF-BP-00148.

Results

To find another source of the pluripotent cells in adult tissue, wemonitored stem cell-specific gene expression (Oct-4, Nanog and Cripto)in the brain, heart, lung, liver, stomach, kidney, ovary, smallintestine, skin, and spleen of 8-week-old, adult female mice. All organsexpressed Nanog mRNA, which was confirmed by RT-PCR (primer sequenceslisted in Table 8), and most organs except for the stomach and skinexpressed Cripto (FIG. 15).

TABLE 8 Oligonucleotide primers and PCR cycling conditions GenBankPrimer sequence Size Temp Genes number Sense (5′ > 3′) Anti-sense(5′ > 3′) (bp) (° C.) β-actin (RT) X03672 ACCGTGAAAAGATGACTCTCAGCTGTGGTGGTG 254 60 CCAG AAG β-actin (R-T) X03672 TACCACAGGCATTGTGATCTTTGATGTCACGCACG 200 60 TGG ATT Oct-4 (RT, R-T) M34381GAAGCCCTCCCTACAGC CAGAGCAGTGACGGGAA 297 60 AGA CAG Nanog (RT, R-T)AY455282 CCCCACAAGCCTTGGAA CTCAAATCCCAGCAACC 255 60 TTA ACA Rex-1 (RT)M28382 ACATCCTAACCCACGCA TGATTTTCTGCCGTATGC 294 60 AAG AA Rex-1 (R-T)M28382 TCCCCGTGTAACATACA CTTCGTCCCCTTTGTCAT 247 60 CCA GT Cripto (RT)M87321 CTTTAAGCAGGGAGGT TAAAGCCATCTGCCACA 195 60 GGTG ATG Cripto (R-T)M87321 CGGAGATCTTGGCTGCT CTTCGACGGCTCGTAAA 200 60 AAC AAC Dnmt3b (RT)BC105922 AGTCCATCGCTGTGGGA GGGCGGGTATAATTCAGC 226 60 ACT AA Dnmt3b (R-T)BC105922 GTCCGGAAAATCACCA CCAGAAGAATGGACGGT 201 60 AGAA TGT Tert (RT)AF051911 GGATCCTGGCTACGTTC TGCCTGACCTCCTCTTGT 208 60 CTG GA Tert (R-T)AF051911 GCAGTGGTCCGGAGAG ACACTGTGACGCAGGAA 224 60 ATAG GTG Lif Rc (RT,R-T) BC031929 GCTGAGTGGTAAAGATA TTCGTTGGACTCATACAA 261 60 CCG CA Stat3(RT) AY299489 TTTGGAATGAAGGGTAC CAAATGACATGTTGTTCA 228 60 ATC GC Bmp4(RT) BC013459 TGAGAGACCCCAGCCT AAACTTGCTGGAAAGGC 259 60 AAGA TCA Fgf4(RT) BC104312 CAGTCTTCTGGAGCTCT AGGAAGTGGGTTACCTT 282 60 CTC CAT Foxd3(RT) AF067421 CAAGAACAGCCTGGTG GTCCAGGGTCCAGTAGT 262 60 AAG TG Sox2 (RT)AB108673 ACGCTCATGAAGAAGG GTAGGACATGCTGTAGG 345 60 ATAA TGG CD9 (RT)U60473 ATGCTACCACTGTTTCC ACAAGTTAAACTGGCAG 212 60 AAC CAT Gdf3 (RT)BC101963 CGAGTTTCAAGACTCTG TAGAGGACCTTCTGGAG 276 60 ACC ACA Zfy1 (gDNA)AC163622 GTTACTCATTTTCAGGT GTGTCAGCTGTTATAGGA 572 62 GTTCTGGG TCAGTGAXist (gDNA) AJ421479.1 GAGATACATTTATTTGCT GACTTAGTTTGGTTTCTT 540 55 CATA RT = Reverse Transcriptase Polymerase Chain Reaction, R-T = Real-TimePolymerase Chain Reaction, gDNA = genomic DNA Polymerase Chain Reaction.

However, the expression of Oct-4 was detected only in the ovary, smallintestine and spleen. In addition to Oct-4, Nanog and Cripto, ovariantissue further expressed the Rex-1, Dnmt3b, Tert, and Lif Rc genes.Their expression levels, however, were different among the ovariesexamined and Nanog expression was even negligible in one ovary (FIG.16).

A very few of Oct-4-positive cells were dissociated from the spleen andsmall intestine, but they were negative for Nanog (FIG. 17). Theseresults demonstrated that Nanog mRNA was not translated in thesetissues. In contrast, the cells dissociated from the ovary were positivefor both Oct-4 and Nanog. Subsequently, we dissociated small intestinal,splenal and ovarian cells by collagenase I and trypsin treatments andcultured them on mouse embryonic fibroblast (MEF) monolayer with orwithout mitomycin-C treatment in Dulbecco's minimal essential medium(DMEM)-based medium containing β-mercaptoethanol, non-essential aminoacids, L-glutamine, fetal bovine serum and antibiotics, to which 1,000,2,000 or 5,000 units/ml leukemia inhibitory factor (LIF) was added.Splenal and small intestinal cells dominantly consisted of fibroblastsand only formed homogenous monolayer without colony-formation,regardless of LIF concentrations. As the control, culture of MEFs alonein the same medium did not yield colonies.

In the case of the ovarian cell culture, dissociated cells were furtherfiltered through a cell strainer before culture for removing immature,maturing and mature oocytes, and most preantral and antral follicles ofmore than 40-μm in diameter. To further remove stromal cells mixed inthe dissociated cells, the buoyant cells above the bottomed cells werecollected 30 min after seeding and reseeded on the MEF monolayer.Colony-like cell aggregation was observed during primary culture, whileMEF-free culture did not yield the colony-formation. The same techniquewas applied for culturing splenal and small intestinal cells, but failedto yield colonies. Two of 14 trials gave colonies until day 7 of primaryculture and all cases of the establishment were observed only afterculture in high dose (5,000 units/ml) LIF-containing medium. Thecolony-forming cells were morphologically typical of embryonic stem (ES)cells or cultured epiblast cells (FIG. 18), which have stably beenmaintained for more than 3 months with 25 passages.

To trace the origin of colony-forming cells, short-tandem repeat (STR)microsatellite analysis using two markers was undertaken. The genotypesof two lines established (OSC-B6D2-SNU-1 and OSC-B6D2-SNU-2), theovarian cell donor (B6D2F1; C57BL/6×DBA2) and the feeder fibroblasts(ICR) were compared. The microsatellite loci of the established cellswere exactly matched with those of the ovary donor, while completelydifferent from those of the fibroblast donors (Table 9).

TABLE 9 Short-tandom repeat microsatellite analysis of establishedcolony-forming cells derived from the coculturing ovarian cells andembryonic fibroblast feeder, and the strains of the ovary (B6D2F1) andfeeder cell (ICR) donor Sample Size 1 Size 2 D3Mit200^(a) Ovary donor101.09 124.22 Feeder cell 124.24 126.21 OSC-B6D2-SNU-1 100.96 124.11OSC-B6D2-SNU-2 100.95 124.19 D11Mit4^(a) Ovary donor 248.92 285.39Feeder cell 242.8 248.98 OSC-B6D2-SNU-1 248.97 285.44 OSC-B6D2-SNU-2249.07 285.53 ^(a)Microsatellite markers used were selected from MITdatabase for discerning mouse strains employed as ovary donor (B6D2F1)and feeder cells (ICR).

Next, in situ-hybridization of ovarian tissue was conducted and strongexpression of stem cell-specific, Oct-4 and Nanog mRNA expression wasdetected predominantly in ovarian medulla and at the thin layer of theperipheral region in preantral and antral follicles. Subsequently,dissociated ovarian cells after stromal cell removal were double stainedwith Nanog, Vasa (germ cell-specific), AMH (follicle cell-specific) orCD44 (mesenchymal cell-specific) and Oct-4. Nanog-, Vasa- orCD44-positive cells were concomitantly positive for Oct-4, whileAMH-positive cells did not react with Oct-4. The positive cells wereround-type with either fine or coarse marginal line. However, theVasa-positive cells were prominently smaller than others andcolony-forming cells (FIG. 19).

The colony-forming cells subpassaged twenty times were positive foralkaline phosphatase, anti-stage specific embryonic antigen (SSEA)-1,anti-integrin α6, anti-integrin β1 and Oct-4 antibodies staining,whereas no reactivity to anti-SSEA-3 or anti-SSEA-4 antibodies wasdetected (FIG. 20). The stem cell-specific Oct-4, Nanog, Rex-1, Cripto,Dnmt3b, Tert, Lif Rc, Stat3, Bmp4, Fgf4, Foxd3, Sox2, CD9, and Gdf3genes were also expressed and telomerase activity was detected in bothlines established (FIG. 21). They had diploid karyotype with XX sexchromosome, which was determined by G-banding of air-dried chromosome,fluorescent-activated cell sorting (FACS) using a flow cytometry and PCRanalysis using X chromosome-specific Xist and Y chromosome-specific Zfy1gene primers (FIG. 22). Neither the established cells nor the referencedE14 ES cells were positive for tissue-specific stem cell markers (Sca-1and CD44 for mesenchymal stem cell, CD34 for epithelial stem cell andCD45 for hematopoietic stem cell, Fragilis and Vasa for germline stemcell) and AMH (FIGS. 23-24).

After culture in LIF-free medium, the cells formed embryoid bodies (FIG.25) consisted of the cells being positive for markers of three germlayers (S-100, Nestin, α-smooth muscle actin, desmin, α-fetoprotein andtroma-1). After being subcutaneously transplanted into NOD-SCID mice,the colony-forming cells formed teratomas consisting of cells derivedfrom three germ layers (neuroepithelial rosettes, keratinized stratifiedsquamous epithelial cells, osteoid island showing bony differentiation,muscle, pancreatic tissue, and ciliated columnar epithelial cells forOSC-B6D2-SNU-1 represented in FIG. 25). The colony-forming cells furtherdifferentiated into neuronal cells (neuron, oligodendrocyte andastrocyte) in an inducible manner after being treated with N2B27solution (FIG. 26). These results show the established have ES cell(ESC)-like activity.

We are now undertaking a final set of experiment to confirm thepluripotency of the established cells by producing germline chimeras,which will be further expanded into a full-scale experiment with usingEGFP-transfected cells (unpublished data). To date, we have had onedelivery after transfer of embryos being aggregated with thecolony-forming cells (OSC-B6D2-SNU-1) retrieved at the 11^(th)subpassage and among 4 live offsprings, one was somatic chimera.

From these series of results, the colony-forming, ESC-like cellsestablished are derived from ovarian cells not derived from feederfibroblasts. Immunohistochemical analysis raised the possibility thatgermline cells (germ cells and parthenogenetic oocytes), follicularcells or mesenchymal cells will be the progenitor cells of theestablished cells. We however exclude germline cells and follicularcells as the progenitor cell and carefully consider the mesenchymal(stem) cells as the progenitor cells. Dissociation procedure consistingof cell strainer filtration can completely eliminate maturing oocytesthat are able to be parthenogenetically activated and growing folliclesof more than 40 μm in diameter. Removing stromal cells and other‘sticky’ cells before seeding minimizes the chance that epithelial orhematopoietic stem cells involve in the establishment. This assumptionis further supported by morphological difference between germlinecell-marker positive cells (apparently smaller) and colony-forming cellsand insensitivity of AMH-positive cells to stem cell-specific marker.The established cells were negative for germline stem cell-specificmarkers as early as they began colony formation and had strongtelomerase expression. Probably, the ‘dormant’ mesenchymal (stem) cellsin the ovary acquire cell plasticity or self-renewal activity, when theyexpose to certain extraordinal environment.

It is apparent that fibroblasts play an important role in establishingovarian ESC-like cells. They may create appropriate niche (e.g.triggering cell-to-cell interaction or secreting critical molecule foracquiring pluripotency) for mobilizing the ‘dormant’ stem cells. Fromdifferent viewpoints, we employed extremely high dose (5,000 units/ml)LIF for the establishment, which would appeal the importance of culturemedium to create/regulate micro-environment for establishingtissue-derived ESC-like cells. In other set of experiment usingdifferent ESC-like cell lines, we found that an increase in Wntsignaling can be triggered by exposure to glutathione, which stimulatedstem cell establishment and maintenance (data not shown).

It is possible that multipotent adult progenitor cells (MAPCs) incirculatory blood are the progenitors of ovarian ESC-like cells. We havenot developed an experimental strategy to determine the progenitor cellof the ESC-like cells between two mesenchymal cells (pluripotent stemcells or MAPCs). However, we hardly consider MAPCs as the progenitorcells, because of tissue specific derivation of the colony-forming cellsonly from the ovaries, and of strong Oct-4 and Nanog expression inovarian medulla (FIG. 19). In fact, most circulatory blood in theovaries is removed during ovarian cell preparation. If MAPCs is theprogenitor of the colony-forming cells, we will have another chance toderive tissue-specific ESC-like cells from various organs. We may derivetissue-specific pluripotent cells by changing culture environment ormedium supplements, while the pluripotent cells can be established byactivating mRNA translation of stem cell-specific genes in certainsomatic cells (FIG. 17).

Our findings present the possibility of establishing autologouspluripotent cells from adult human organs without undertaking SCNT andwithout using embryos and even gametes, which contribute to overcomingthe current limitations of ES cell research. Apparently, thisnewly-suggested alternative for deriving immune-specific pluripotentcells can avoid ethical dispute on undertaking cell/tissue therapy.Cellular and genetic evaluation of the established cells, and comparisonbetween adult tissue-derived ES cells with embryo-derived pluripotentcells can provide numerous cues for further developing novel cell andtissue therapy.

Having described a preferred embodiment of the present invention, it isto be understood that variants and modifications thereof falling withinthe spirit of the invention may become apparent to those skilled in thisart, and the scope of this invention is to be determined by appendedclaims and their equivalents.

REFERENCES

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1. A method for preparing an embryonic stem cell (ESC)-like cell, whichcomprises the steps of: (a) obtaining a first cell population from amammalian tissue or body fluid, wherein the first cell populationcomprises adult stem cells; (b) obtaining a second somatic cellpopulation from a mammalian tissue, wherein the mammalian tissue isdifferent from the mammalian tissue in step (a) and the second cellpopulation is different from the first cell population; (c) coculturingthe first cell population and the second cell population in a medium fora period of time sufficient to form a colony from either the first cellpopulation or the second cell population; and (d) subculturing a cellfrom the colony in a medium for a period time sufficient to prepare theESC-like cell.
 2. The method according to claim 1, wherein the mammaliantissue or body fluid of step (a) is derived from ovary, testis, bonemarrow, peripheral blood, umbilical cord blood, amniotic fluid, brain,blood vessel, skeletal muscle, epithelia of skin or gastrointestinaltract, cornea, dental pulp of tooth, retina, liver, spleen or pancreas.3. The method according to claim 2, wherein the mammalian tissue isderived from ovary.
 4. The method according to claim 1, wherein thefirst cell population is a heterogeneous population comprising at leasttwo cell types.
 5. The method according to claim 1, wherein the secondsomatic cell population is a homogenous population substantiallycomprising one somatic cell type.
 6. The method according to claim 5,wherein the somatic cell is fibroblast or epithelia cell.
 7. The methodaccording to claim 1, wherein the second somatic cell population is amitotically inactive cell.
 8. The method according to claim 1, whereinthe second somatic cell population has adherent characteristics toculture plates.
 9. The method according to claim 1, wherein the firstcell population is obtained from a post-puberty mammalian tissue or bodyfluid.
 10. The method according to claim 1, wherein the first cellpopulation comprising adult stem cells is an ovarian cell.
 11. Themethod according to claim 10, wherein the ovarian cell is substantiallyfreed from ovarian stromal cells.
 12. The method according to claim 10,wherein the ovarian cell is substantially freed from oocytes andpreantral and antral follicles.
 13. The method according to claim 1,wherein the mammal is human, bovine, sheep, ovine, pig, horse, rabbit,goat, mouse, hamster or rat.
 14. The method according to claim 1,wherein the coculturing in the step (c) is carried out in the presenceof at least 3,000 units/ml of leukemia inhibitory factor.
 15. The methodaccording to claim 14, wherein the coculturing in the step (c) iscarried out in the presence of 4,000-6000 units/ml of leukemiainhibitory factor.
 16. The method according to claim 1, wherein thesubculturing in the step (d) is carried out in the presence of 800-1,200units/ml of leukemia inhibitory factor.
 17. The method according toclaim 1, wherein when the ESC-like cell is prepared from the colonyderived from the second cell population, the ESC-like cell has atetraploid karyotype.
 18. The method according to claim 1, wherein whenthe ESC-like cell having tetraploid karyotype is induced todifferentiate, the ESC-like cell has a diploid karyotype.
 19. The methodaccording to claim 1, wherein when the ESC-like cell is prepared fromthe colony derived from the first cell population, the ESC-like cell hasa diploid karyotype.
 20. The method according to claim 1, wherein thecolony is formed from the first cell population having higherenvironmental susceptibility than the second cell population.
 21. Themethod according to claim 1, wherein the colony is formed from thesecond cell population having higher environmental susceptibility thanthe first cell population. 22-43. (canceled)
 44. A mammalian tissue orbody fluid-derived embryonic stem cell (ESC)-like cell, wherein theESC-like cell has pluripotency; the ESC-like cell is prepared bycoculturing (i) an adult stem cell-containing first cell population froma mammalian tissue or body fluid and (ii) a second somatic cellpopulation from a mammalian tissue different from the mammalian tissueof (i); and the ESC-like cell is not prepared by a somatic cell nucleartransfer.
 45. The ESC-like cell according to claim 44, wherein themammalian tissue or body fluid for the stem cell-containing first cellpopulation is derived from ovary, bone marrow, peripheral blood,umbilical cord blood, amniotic fluid, brain, blood vessel, skeletalmuscle, epithelia of skin or gastrointestinal tract, cornea, dental pulpof tooth, retina, liver, spleen or pancreas.
 46. The ESC-like cellaccording to claim 45, wherein the mammalian tissue is derived fromovary.
 47. The ESC-like cell according to claim 44, wherein the firstcell population is a heterogeneous population comprising at least twocell types.
 48. The ESC-like cell according to claim 44, wherein thesecond somatic cell population is a homogenous population substantiallycomprising one somatic cell type.
 49. The ESC-like cell according toclaim 48, wherein the somatic cell is fibroblast or epithelia cell. 50.The ESC-like cell according to claim 44, wherein the second somatic cellpopulation is a mitotically inactive cell.
 51. The ESC-like cellaccording to claim 44, wherein the second somatic cell population hasadherent characteristics to culture plates.
 52. The ESC-like cellaccording to claim 44, wherein the first cell population is obtainedfrom a post-puberty mammalian tissue or body fluid.
 53. The ESC-likecell according to claim 44, wherein the first cell population comprisingstem cells is an ovarian cell.
 54. The ESC-like cell according to claim53, wherein the ovarian cell is substantially freed from ovarian stromalcells.
 55. The ESC-like cell according to claim 53, wherein the ovariancell is substantially freed from oocytes and preantral and antralfollicles.
 56. The ESC-like cell according to claim 44, wherein themammal is human, bovine, sheep, ovine, pig, horse, rabbit, goat, mouse,hamster or rat.
 57. The ESC-like cell according to claim 44, whereinwhen the ESC-like cell is originated from the second cell population,the ESC-like cell has a tetraploid karyotype.
 58. The ESC-like cellaccording to claim 57, wherein when the ESC-like cell with tetraploidkaryotype is induced to differentiate, the ESC-like cell has a diploidkaryotype.
 59. The ESC-like cell according to claim 60, wherein when theESC-like cell is originated from the first cell population, the ESC-likecell has a diploid karyotype.
 60. The ESC-like cell according to claim44, wherein when the ESC-like cell is originated from the first cellpopulation, the ESC-like cell shows a positive reactivity to alkalinephosphatase, and to an antibody against each of stage specific embryonicantigen (SSEA)-1, integrin α6, integrin β1, and Oct-4, and a negativereactivity to an antibody against each SSEA-3 and SSEA-4.
 61. TheESC-like cell according to claim 60, wherein the ESC-like cell expressesat least one stem-cell specific gene selected from the group consistingof Oct-4, Nanog, Rex-1, Cripto, Dnmt3b, Tert, Lif Rc, Stat3, Bmp4, Fgf4,Foxd3, Sox2, CD9, and Gdf3.
 62. The ESC-like cell according to claim 60,wherein the ESC-like cell shows a negative reactivity to an antibodyagainst Sca-1, CD44, CD34, CD45, Fragilis, Vasa and/or AMH(anti-mullerian hormone).
 63. The ESC-like cell according to claim 44,wherein when the ESC-like cell is originated from the second cellpopulation, the ESC-like cell shows a positive reactivity to alkalinephosphatase, and to an antibody against each of stage specific embryonicantigen (SSEA)-1, integrin α6, integrin β1 and Oct-4, and a negativereactivity to an antibody against each SSEA-3 and SSEA-4. 64-80.(canceled)